Research on Cell Cycle and DNA Damage Response Mechanisms

The cell cycle is an ordered process through which cells complete DNA replication, chromosome segregation, and cell division. The DNA damage response (DDR) is responsible for pausing the cell cycle and initiating repair when replication errors, DNA breaks, or replication stress occur.

Keywords: cell cycle; DNA damage response; G1/S checkpoint; G2/M checkpoint; ATM; ATR; Chk1; Chk2; CDK; genome stability

1 Basic Logic of Cell Cycle and DNA Damage Research

1.1 Phase Control of the Cell Cycle

The cell cycle usually proceeds in the order of G1–S–G2–M. S phase is responsible for DNA replication, M phase is responsible for chromosome segregation and cell division, while G1 and G2 phases provide buffer windows for cells to assess nutritional status, external signals, DNA integrity, and the completion of replication.

(1) G1 phase

G1 phase determines whether a cell enters the proliferative program. Cells need to integrate signals related to growth factors, nutritional status, cell size, DNA damage, and extracellular stress. If the conditions are sufficient, CDK4/6-cyclin D and CDK2-cyclin E are gradually activated, driving the cell into S phase.

(2) S phase

S phase carries out whole-genome DNA replication. This phase is highly vulnerable to replication stress, such as replication fork stalling, nucleotide insufficiency, DNA template damage, and replication-transcription conflicts. Abnormalities during S phase can directly trigger the ATR-Chk1 axis.

(3) G2 phase

G2 phase is used to confirm whether DNA replication has been completed, whether DNA damage has been repaired, and whether chromosomes are ready to enter mitosis. If double-strand breaks or incomplete replication are present, cells should remain in G2 phase to avoid premature entry into M phase.

(4) M phase

M phase completes chromosome condensation, spindle formation, chromosome alignment, and sister chromatid separation. This phase mainly depends on Cdk1-cyclin B, Aurora kinases, PLK1, and the spindle assembly checkpoint.

1.2 Role of Cell Cycle Checkpoints

Cell cycle checkpoints are not simple “switches,” but quality control systems that determine whether cells meet the conditions required to enter the next phase. Their core functions include:

(1) Preventing damaged DNA from being replicated or segregated;

(2) Buying time for DNA repair;

(3) Preventing incompletely replicated chromosomes from entering mitosis;

(4) Inducing senescence, apoptosis, or other cell fate changes when damage cannot be repaired;

(5) Limiting the expansion of genomic instability.

Table 1 Cell Cycle Phases and Major Regulatory Nodes

Phase	Core Event	Major Regulatory Molecules	Research Focus
G1 phase	Integration of growth signals and passage through the restriction point	cyclin D-CDK4/6, pRB, E2F, p21, p27	Proliferation initiation, G1/S transition, abnormal tumor cell cycle control
S phase	DNA replication and replication fork progression	cyclin E/A-CDK2, ATR, Chk1, RPA, PCNA	Replication stress, replication fork stability, DNA synthesis rate
G2 phase	Replication completion check and DNA damage repair	ATR, ATM, Chk1, Chk2, Wee1, Cdc25C	G2/M arrest, damage repair, Cdk1 inhibition
M phase	Chromosome segregation and cell division	cyclin B-CDK1, PLK1, Aurora, APC/C	Mitotic entry, spindle checkpoint, chromosome stability

 Figure 1 Schematic diagram of cell-cycle phase transition and DNA damage checkpoint regulatory networks

2 G1/S Checkpoint and Proliferation Initiation

2.1 pRB-E2F Axis

(1) Mechanistic core

The G1/S transition is mainly controlled by the pRB-E2F axis. Unphosphorylated or hypophosphorylated pRB can bind E2F transcription factors and suppress the expression of S phase-related genes. After CDK4/6-cyclin D and CDK2-cyclin E progressively phosphorylate pRB, E2F is released, promoting the expression of DNA replication-related genes and driving the cell into S phase.

(2) Experimental indicators

Common indicators include pRB phosphorylation, E2F target gene expression, cyclin D, cyclin E, CDK4/6, CDK2, p21, p27, EdU incorporation, BrdU incorporation, and flow cytometric cell cycle analysis.

(3) Result interpretation

Increased pRB phosphorylation usually suggests release of the G1/S restriction point, but S phase proportion and DNA synthesis indicators should also be combined for confirmation. If pRB phosphorylation increases but EdU incorporation does not increase, replication stress, nutritional limitation, or downstream replication initiation defects may be present.

2.2 CDK Inhibitors

(1) Mechanistic core

CDK inhibitors such as p21 and p27 can inhibit CDK activity, restrict pRB phosphorylation, and limit S phase entry. After p53 activation, p21 expression can be induced, thereby connecting DNA damage signaling with G1 arrest.

(2) Experimental indicators

p53, p-p53, p21, p27, CDK activity, pRB phosphorylation, and the proportion of G1-phase cells are commonly detected.

(3) Result interpretation

Increased p21 together with decreased pRB phosphorylation and an increased G1-phase proportion supports G1/S arrest. If p21 increases but cells still enter S phase, pRB loss, excessive CDK activation, or checkpoint failure in tumor cells should be considered.

2.3 G1 Checkpoint Defects and Tumors

(1) Mechanistic core

Many tumor cells have p53 mutations, pRB inactivation, cyclin D amplification, or CDK4/6 hyperactivation, allowing cells to enter S phase even under DNA damage conditions. This defect can increase replication stress and mutational burden, while also creating therapeutic vulnerabilities.

(2) Experimental indicators

TP53, RB1, CCND1, CDK4/6, E2F target genes, S phase proportion, DNA damage markers, and sensitivity to CDK inhibitors can be detected.

(3) Result interpretation

If tumor cells depend on CDK4/6 to maintain pRB phosphorylation, CDK4/6 inhibitors may induce G1 arrest. If RB1 is lost, CDK4/6 inhibitors usually have difficulty producing typical arrest through the pRB-E2F axis.

3 G2/M Checkpoint and Mitotic Entry

3.1 Cdk1-Cyclin B Activation

(1) Mechanistic core

The G2/M transition depends on activation of cyclin B-Cdk1. Cdk1 remains inhibited after phosphorylation by Wee1, while dephosphorylation of Cdk1 by Cdc25C promotes its activation. PLK1 can promote Cdc25 activation and inhibit Wee1, thereby driving cells into mitosis.

(2) Experimental indicators

Commonly detected indicators include cyclin B1, Cdk1 Tyr15 phosphorylation, Wee1, p-Wee1, Cdc25C, p-Cdc25C, PLK1, p-Histone H3, and M phase proportion.

(3) Result interpretation

Increased Cdk1 Tyr15 phosphorylation usually indicates G2/M arrest, whereas increased p-Histone H3 indicates increased mitotic entry. If Cdk1 remains activated after DNA damage, the G2/M checkpoint may have failed, creating the risk that cells enter M phase with damaged DNA.

3.2 ATR-Chk1-Wee1/Cdc25C Axis

(1) Mechanistic core

When DNA replication is abnormal or replication forks stall, ATR is activated and further activates Chk1. Chk1 can inhibit Cdc25C, and ATR can also maintain Cdk1 inhibition by enhancing Wee1 function, ultimately preventing premature activation of cyclin B-Cdk1.

(2) Experimental indicators

p-ATR, p-Chk1, Wee1, p-Cdc25C, Cdk1 Tyr15 phosphorylation, γH2AX, RPA32 phosphorylation, and cell cycle distribution are commonly detected.

(3) Result interpretation

The simultaneous appearance of increased p-Chk1, inhibited Cdc25C, and increased Cdk1 Tyr15 phosphorylation supports ATR-Chk1-mediated G2/M arrest. Detection of γH2AX elevation alone cannot directly indicate whether the checkpoint is effective.

3.3 ATM-Chk2 and Double-Strand Break Response

(1) Mechanistic core

Double-strand DNA breaks (DSBs) can activate ATM. ATM transmits damage signals through Chk2, p53, H2AX, and multiple repair factors, and can affect the G1/S, S phase, and G2/M checkpoints simultaneously.

(2) Experimental indicators

p-ATM, p-Chk2, γH2AX, 53BP1 foci, p-p53, p21, cell cycle distribution, and dynamics of DNA repair foci are commonly detected.

(3) Result interpretation

Increased p-ATM and γH2AX indicate activation of a DSB-related response, but whether repair has been completed should be evaluated by observing the disappearance of γH2AX/53BP1 foci, recovery of the cell cycle, and clonogenic capacity. Persistent damage markers usually indicate insufficient repair or damage accumulation.

Table 2 Core Mechanisms of G1/S and G2/M Checkpoints

Checkpoint	Main Trigger	Core Molecules	Main Outcome
G1/S checkpoint	DNA damage, insufficient growth signals, p53 activation	p53, p21, CDK4/6, pRB, E2F	Prevents cells from entering S phase
S phase checkpoint	Replication fork stalling, replication stress, single-stranded DNA accumulation	ATR, Chk1, RPA, Claspin	Reduces replication initiation and stabilizes replication forks
G2/M checkpoint	DSBs, incomplete replication, ATR/ATM activation	Chk1, Chk2, Wee1, Cdc25C, Cdk1	Prevents cells from entering mitosis
Spindle checkpoint	Chromosomes not properly attached to the spindle	MAD2, BUBR1, APC/C, Aurora	Prevents erroneous chromosome segregation

4 Recognition and Signal Transduction in the DNA Damage Response

4.1 Basic Components of DDR

The DNA damage response includes damage recognition, signal amplification, cell cycle arrest, DNA repair, replication restart, and damage bypass. Its purpose is not merely to “repair DNA,” but to maintain genome stability under damaged conditions and determine cell fate.

(1) Damage recognition

DNA double-strand breaks, exposed single-stranded DNA, replication fork stalling, and base damage can be recognized by different sensor systems. For example, the MRN complex participates in DSB recognition, while RPA-coated single-stranded DNA is an important signal for ATR activation.

(2) Signal amplification

PIKK family kinases such as ATM, ATR, and DNA-PK can phosphorylate a large number of substrates, forming a damage signaling network. γH2AX can form chromatin marks around damage sites and promote recruitment of repair proteins.

(3) Effector output

DDR can induce cell cycle arrest, DNA repair, replication fork protection, transcriptional regulation, senescence, apoptosis, or mitotic catastrophe. Different outputs depend on damage type, damage intensity, repair capacity, and cellular context.

4.2 ATM Pathway

(1) Mechanistic core

ATM mainly responds to DNA double-strand breaks. After DSBs occur, the MRN complex helps localize and activate ATM. ATM further phosphorylates substrates such as H2AX, Chk2, p53, and KAP1, promoting damage signal spreading and repair factor recruitment.

(2) Experimental indicators

p-ATM, γH2AX, p-Chk2, p-p53, 53BP1 foci, MRE11/RAD50/NBS1 localization, and DSB repair dynamics are commonly detected.

(3) Result interpretation

ATM activation indicates that cells have recognized DSBs or similar damage stress, but it alone does not show that repair has been completed. If γH2AX and 53BP1 foci do not disappear for a long time, persistent damage or insufficient repair is suggested.

4.3 ATR Pathway

(1) Mechanistic core

ATR mainly responds to replication stress and single-stranded DNA accumulation. When replication forks stall, uncoupling between DNA unwinding and synthesis can generate RPA-coated single-stranded DNA, thereby promoting ATR-Chk1 pathway activation, inhibiting new origin firing, and stabilizing existing replication forks.

(2) Experimental indicators

p-ATR, p-Chk1, RPA32 phosphorylation, replication fork speed, EdU incorporation, DNA fiber assay, and S phase proportion are commonly detected.

(3) Result interpretation

Increased p-Chk1 and RPA32 phosphorylation indicate enhanced replication stress. If EdU decreases and γH2AX increases simultaneously, replication blockage may have already converted into DNA breaks or replication fork collapse.

4.4 DNA-PK Pathway

(1) Mechanistic core

DNA-PK mainly participates in non-homologous end joining (NHEJ). After Ku70/Ku80 recognize DNA ends, DNA-PKcs is recruited to promote end protection, processing, and ligation.

(2) Experimental indicators

DNA-PKcs phosphorylation, Ku70/Ku80 recruitment, NHEJ reporter systems, DSB repair efficiency, and radiosensitivity are commonly detected.

(3) Result interpretation

DNA-PK activation often suggests involvement of NHEJ in DSB repair. If DNA-PK inhibition enhances sensitivity to radiotherapy or DSB-inducing agents, tumor cells may depend on NHEJ for survival.

5 DNA Repair Pathways and Damage Types

5.1 Homologous Recombination Repair

(1) Mechanistic core

Homologous recombination (HR) uses sister chromatids as templates for high-fidelity repair and mainly occurs in S and G2 phases. BRCA1, BRCA2, and RAD51 are key molecules in the HR pathway.

(2) Experimental indicators

RAD51 foci, BRCA1/2 status, HR reporter systems, γH2AX disappearance, PARP inhibitor sensitivity, and platinum drug sensitivity are commonly detected.

(3) Result interpretation

Insufficient RAD51 foci formation indicates HR deficiency. BRCA1/2-deficient cells are sensitive to PARP inhibitors, reflecting a synthetic lethality relationship, but HR functional testing should still be combined for confirmation.

5.2 Non-Homologous End Joining

(1) Mechanistic core

NHEJ does not depend on homologous templates and can repair DSBs in multiple cell cycle phases, especially in G1 phase. It is relatively fast, but may introduce small insertions or deletions.

(2) Experimental indicators

Ku70/Ku80, DNA-PKcs, XRCC4, Ligase IV, NHEJ reporter systems, and chromosomal breaks or rearrangements can be detected.

(3) Result interpretation

Enhanced NHEJ can help cells rapidly repair breaks, but in the context of error-prone repair it may also increase chromosomal rearrangements. In tumor cells, increased NHEJ dependence may be associated with radiotherapy tolerance.

5.3 Base Excision Repair, Nucleotide Excision Repair, and Mismatch Repair

(1) BER

Base excision repair (BER) mainly processes oxidized bases, alkylated bases, and single-strand breaks, often involving PARP1, XRCC1, APE1, and DNA polymerase β.

(2) NER

Nucleotide excision repair (NER) mainly processes ultraviolet-induced thymine dimers and bulky DNA adducts, often involving molecules such as XPA, XPC, ERCC1, and XPF.

(3) MMR

Mismatch repair (MMR) is responsible for repairing post-replication base mismatches and small insertion/deletion loops, commonly involving MSH2, MSH6, MLH1, and PMS2. MMR deficiency can lead to microsatellite instability.

(4) Result interpretation

Different repair pathways correspond to different damage types, and γH2AX alone should not be used to summarize all DNA damage. For oxidative damage studies, 8-OHdG and BER should be emphasized; for UV damage studies, CPD and NER should be emphasized; for replication error studies, MMR status and microsatellite instability should be emphasized.

Table 3 DNA Damage Types and Major Repair Pathways

Damage Type	Representative Damage	Main Repair Pathway	Key Molecules
Double-strand breaks	Radiotherapy, topoisomerase inhibitors, replication fork collapse	HR, NHEJ	BRCA1/2, RAD51, Ku70/80, DNA-PKcs
Single-strand breaks	Oxidative stress, alkylation damage	BER	PARP1, XRCC1, APE1
UV damage	CPD, 6-4PP	NER	XPA, XPC, ERCC1, XPF
Replication mismatch	Base mismatch, small insertion/deletion loops	MMR	MSH2, MSH6, MLH1, PMS2
Replication stress	Replication fork stalling, single-stranded DNA accumulation	ATR-Chk1, HR	ATR, Chk1, RPA, BRCA1, RAD51

6 Replication Stress and Genomic Instability

6.1 Sources of Replication Stress

Replication stress refers to obstacles during DNA replication that cause abnormal replication fork progression or delayed replication completion. Tumor cells often experience persistent replication stress due to oncogene activation, excessive origin firing, nucleotide insufficiency, transcription-replication conflicts, and DNA template damage.

(1) Oncogene activation

MYC, RAS, cyclin E, and related factors can promote replication initiation and accelerate the cell cycle, increasing the burden on the replication system.

(2) Nucleotide insufficiency

Insufficient dNTPs reduce DNA synthesis efficiency and cause replication fork stalling.

(3) Transcription-replication conflicts

When highly transcribed regions encounter replication forks, R-loop formation and replication fork instability can increase.

(4) DNA template damage

Oxidative damage, crosslink damage, or abnormal DNA topology can hinder polymerase progression.

6.2 Replication Fork Protection and Collapse

(1) Mechanistic core

Mild replication stress can maintain replication fork stability through ATR-Chk1, inhibit excessive replication origin firing, and buy time for repair. If stress persists, replication forks may collapse and convert into double-strand breaks.

(2) Experimental indicators

DNA fiber assay is commonly used to detect replication fork speed, replication fork stalling, and replication restart. RPA, p-RPA32, p-Chk1, γH2AX, RAD51, and EdU incorporation can also be detected.

(3) Result interpretation

Reduced replication fork speed indicates increased replication stress. Increased RPA and p-Chk1 indicate activation of the ATR pathway. Increased γH2AX suggests that replication stress may have already caused DNA breaks.

6.3 Replication Stress Dependence of Tumor Cells

(1) Mechanistic core

Tumor cells often have high replication stress and depend on checkpoints such as ATR, Chk1, and Wee1 for survival. Inhibition of these pathways can force tumor cells with unrepaired DNA to enter mitosis, inducing mitotic catastrophe.

(2) Experimental indicators

p-Chk1, Wee1, Cdk1 Tyr15 phosphorylation, γH2AX, p-Histone H3, cell death, clonogenic formation, and selective killing of tumor cells can be detected.

(3) Result interpretation

If γH2AX and p-Histone H3 increase simultaneously after ATR, Chk1, or Wee1 inhibition, cells may be entering M phase before DNA damage is repaired, indicating checkpoint override and a risk of mitotic catastrophe.

7 Cell Cycle/DDR Targets and Tumor Therapy

7.1 CDK Targets

(1) Mechanistic core

CDKs drive cell cycle progression. CDK4/6 mainly controls the G1/S restriction point, CDK2 participates in S phase entry and replication regulation, and CDK1 controls the G2/M transition and mitotic entry. Abnormal CDK activity in tumor cells can drive uncontrolled proliferation.

(2) Research indicators

pRB, E2F target genes, cyclin D/E/A/B, CDK activity, EdU incorporation, cell cycle distribution, and clonogenic formation are commonly detected.

(3) Result interpretation

Whether CDK inhibitors cause G1 arrest or G2/M arrest depends on the targeted CDK type and cellular context. The effect of CDK4/6 inhibition usually depends on functional pRB, and drug sensitivity should be interpreted cautiously when RB1 is lost.

7.2 ATR, Chk1, and Wee1 Targets

(1) Mechanistic core

ATR, Chk1, and Wee1 help cells cope with replication stress and DNA damage. If tumor cells have p53 defects or high replication stress, they may depend more strongly on the G2/M checkpoint and therefore be more sensitive to ATR, Chk1, or Wee1 inhibition.

(2) Research indicators

p-Chk1, p-RPA32, Cdk1 Tyr15 phosphorylation, γH2AX, p-Histone H3, cell death, and clonogenic formation can be detected.

(3) Result interpretation

If inhibition of ATR/Chk1/Wee1 increases DNA damage and is accompanied by increased M phase entry, checkpoint release is suggested. At this point, cell death may result from mitotic catastrophe rather than simple apoptosis.

7.3 PARP and Synthetic Lethality

(1) Mechanistic core

PARP participates in single-strand break repair. HR-deficient cells, especially BRCA1/2-deficient cells, are more sensitive to PARP inhibition because accumulated single-strand breaks can be converted into double-strand breaks during replication, while cells lack effective HR repair capacity.

(2) Research indicators

PAR levels, γH2AX, RAD51 foci, BRCA1/2 status, HR function, cell survival, and clonogenic formation are commonly detected.

(3) Result interpretation

Sensitivity to PARP inhibitors cannot be inferred only from BRCA mutations; HR functional status should also be considered. If RAD51 foci are restored, HR function may have recovered, leading to PARP inhibitor resistance.

7.4 Aurora Kinases and PLK1

(1) Mechanistic core

Aurora kinases and PLK1 regulate mitotic entry, chromosome alignment, centrosome function, spindle formation, and cytokinesis. Their abnormal activation can promote chromosomal instability and represents an important direction for mitosis-targeted therapy in tumor cells.

(2) Research indicators

p-Histone H3, spindle morphology, chromosome misalignment, polyploidy, failed cell division, PLK1 activity, and phosphorylation of Aurora substrates are commonly detected.

(3) Result interpretation

Mitosis-targeting drugs often cause M phase arrest, abnormal chromosome segregation, or polyploidy. If the final mode of cell death is unclear, apoptosis, senescence, mitotic slippage, and mitotic catastrophe should be distinguished.

Table 4 Therapeutic Targets Related to Cell Cycle/DDR

Target Category	Representative Molecules	Action Stage	Research Significance
CDK	CDK4/6, CDK2, CDK1	G1/S, S phase, G2/M transition	Inhibits tumor cell cycle progression
Checkpoint kinases	ATR, Chk1, Chk2, Wee1	Replication stress and G2/M checkpoint	Induces checkpoint override and mitotic catastrophe
DNA repair factors	PARP1, BRCA1/2, RAD51, DNA-PK	DNA repair	Synthetic lethality and sensitization to radiotherapy/chemotherapy
Mitotic kinases	Aurora, PLK1	M phase progression and chromosome segregation	Inhibits mitosis and induces chromosomal instability
p53-p21 axis	p53, p21	G1/S arrest and post-damage fate determination	Evaluates checkpoint integrity and therapeutic response

Table 5 Product Lines Related to Cell Cycle/DNA Damage and Application Directions

Research Direction	Product Line	Covered Object/Target	Applicable Research Direction
DNA damage induction research	DNA Alkylator/Crosslinker	Reagents related to alkylation damage, DNA crosslinking, and replication blockade	DNA adducts, replication fork stalling, DDR activation, and chemotherapy mechanism research
DNA synthesis blockade research	Nucleoside Antimetabolite/Analog	Nucleoside analogs and antimetabolite drugs	S-phase arrest, replication stress, DNA synthesis inhibition, and antitumor efficacy research
Folate metabolism and nucleic acid synthesis research	Antifolate	Folate metabolism, dTMP synthesis, and nucleotide supply	DNA synthesis limitation, S-phase arrest, replication stress, and antimetabolite drug research
DNA/RNA synthesis research	DNA/RNA Synthesis	Substrates, inhibitors, and detection tools related to nucleic acid synthesis	DNA replication, RNA transcription, nucleic acid synthesis rate, and replication stress research
DNA staining and cell cycle detection	DNA Stain	DNA content staining and nucleic acid labeling tools	PI flow cytometry, cell cycle distribution, nuclear morphology, and DNA content detection
DNA structure research	G-quadruplex	G-quadruplex structures and related stabilizing tools	Telomeres, replication stress, transcriptional regulation, and genome stability research
DNA break and repair research	ATM/ATR	ATM, ATR, and related signaling pathways	DSB response, replication stress, checkpoint activation, and DDR signal transduction research
Checkpoint kinase research	Checkpoint Kinase (Chk)	Chk1, Chk2, and other checkpoint kinases	G1/S, S-phase, and G2/M checkpoints, replication stress, and DNA damage arrest research
G2/M checkpoint research	Wee1	Wee1 kinase and Cdk1 inhibitory regulation	G2/M arrest, Cdk1 Tyr15 phosphorylation, checkpoint abrogation, and mitotic catastrophe research
DNA double-strand break repair research	DNA-PK	DNA-PKcs and NHEJ-related targets	Non-homologous end joining, radiosensitization, DSB repair, and DNA-PK inhibitor research
Homologous recombination repair research	RAD51	RAD51 and HR repair-related molecules	HR function, RAD51 foci, BRCA-related repair defects, and PARP inhibitor sensitivity research
PAR metabolism and DNA repair research	PARP	PARP family members and PAR generation	Single-strand break repair, BER, synthetic lethality, PARP inhibitors, and HR deficiency research
PAR degradation research	Poly(ADP-ribose) Glycohydrolase (PARG)	PARG and PAR chain degradation	PAR dynamics, DNA repair balance, replication stress, and PARP pathway compensation research
Nucleic acid cleavage and damage modeling	Endonuclease	DNA/RNA endonuclease-related tools	DNA break models, repair substrate construction, nucleic acid damage, and genome editing validation
Gene editing and functional validation	CRISPR/Cas9	Cas9 and genome editing systems	DDR gene knockout, cell cycle regulatory gene screening, and functional validation
G1/S transition research	CDK	CDK1/2/4/6 and other cyclin-dependent kinases	G1/S, S-phase, and G2/M transitions, CDK inhibitors, and tumor proliferation research
G1/S transcriptional regulation research	Early 2 Factor (E2F)	E2F family and the pRB downstream transcriptional network	pRB-E2F axis, S-phase gene expression, restriction point passage, and tumor proliferation research
Kinesin-related cell cycle research	Kinesin	Kinesins and microtubule motor proteins	Chromosome movement, spindle dynamics, mitotic progression, and cell division research
Cell cycle-related kinase research	Cyclin G-associated Kinase (GAK)	GAK and cell cycle-related kinase regulation	Cell cycle regulation, membrane trafficking, stress response, and tumor cell adaptability research
Mitotic entry research	Polo-like Kinase (PLK)	PLK1 and other Polo-like kinases	G2/M transition, spindle formation, Cdc25 regulation, and mitosis-targeted therapy
Mitotic kinase research	Aurora Kinase	Aurora A/B/C	Centrosome maturation, chromosome alignment, spindle checkpoint regulation, and chromosomal instability research
Mitotic checkpoint research	Mps1	TTK/Mps1 kinase	Spindle assembly checkpoint, chromosome attachment, and mitotic arrest research
Mitotic regulation research	MASTL	Greatwall/MASTL kinase	Mitotic entry, PP2A inhibition, M-phase maintenance, and cell cycle synchronization research
Chromosome segregation research	Separase	Separase-related targets	Sister chromatid separation, centromere release, and chromosome stability research
Spindle and microtubule research	Microtubule/Tubulin	Tubulin and microtubule-stabilizing or -destabilizing tools	Mitotic spindle, microtubule dynamics, M-phase arrest, and anti-mitotic drug research
Mitotic kinase research	Haspin Kinase	Haspin and H3T3 phosphorylation-related regulation	Chromosome positioning, CPC recruitment, Aurora B regulation, and mitotic chromosome behavior research
Mitotic kinase research	NEKs	NIMA-related kinases	Centrosomes, cilia, spindle formation, and cell cycle progression research
Cytoskeleton and cell cycle migration research	LIM Kinase (LIMK)	LIMK/cofilin axis	Actin remodeling, cell migration, cytokinesis, and tumor cell motility research
Cytoskeletal signaling research	PAK	p21-activated kinases	Cytoskeleton, migration, proliferation, survival, and cell cycle-related signaling research
Rho pathway research	ROCK	RhoA/ROCK pathway	Cell contraction, migration, cytokinesis, cell morphology, and tumor invasion research
Casein kinase-related cell cycle research	Casein Kinase	CK1/CK2 and related kinases	Cyclin stability, DDR regulation, Wnt/cell cycle crosstalk, and protein phosphorylation research
Cell stress and DDR research	HSP	Heat shock proteins	Protein homeostasis, DNA damage stress, tumor cell survival, and therapeutic resistance research
ER stress and cell cycle regulation	ATF6	UPR transcription factor ATF6	ER stress, protein homeostasis, cell cycle arrest, and stress adaptation research
ER stress and cell fate research	IRE1	IRE1/XBP1 pathway	ER stress, cell survival, apoptosis, and DNA damage therapy response research
ER stress and translational control	PERK	PERK/eIF2α pathway	Translational inhibition, stress-induced cell cycle arrest, apoptosis, and tumor tolerance research
Protein folding and stress research	PDI	Protein disulfide isomerase	Protein folding, ER homeostasis, redox stress, and cellular stress response research
Translation initiation and proliferation research	Eukaryotic Initiation Factor (eIF)	eIF family	Protein translation, cell growth, stress-induced translational inhibition, and tumor proliferation research
Ubiquitination regulation research	Deubiquitinase	DUB family	Cyclin stability, DNA repair protein degradation, and DDR signaling regulation research
Protein degradation and stress research	p97	VCP/p97 protein quality control system	Protein degradation, replication stress, chromatin-associated protein clearance, and DDR adaptation research
Chromatin remodeling research	SWI/SNF Complex	SWI/SNF chromatin remodeling factors	DNA repair accessibility, transcriptional regulation, tumor suppression, and chromatin state research
Epigenetic regulation research	HDAC	Histone deacetylases	Chromatin compaction, DNA repair, cell cycle arrest, and HDAC inhibitor research
Deacetylation and stress research	Sirtuin	NAD⁺-dependent deacetylases	DNA repair, metabolic stress, senescence, chromatin homeostasis, and tumor adaptation research
Telomere maintenance research	Telomerase	Telomerase-related targets	Telomere extension, replicative senescence, tumor immortalization, and telomere damage research
DNA topology and breakage research	Topoisomerase	TOP1, TOP2, and other topoisomerases	DNA topological stress, replication/transcription conflict, DSB induction, and chemotherapy mechanism research
Cell death and drug response research	ClpP	Mitochondrial protease ClpP	Mitochondrial protein homeostasis, stress-induced death, tumor metabolism, and drug-induced cell death research
Immune regulation and cell cycle crosstalk	PPAR	Nuclear receptor PPAR family	Metabolism-cell cycle crosstalk, inflammation, tumor metabolism, and cell differentiation research
Molecular chaperone and drug sensitivity research	FKBP	FK506-binding protein family	Protein folding, mTOR-related signaling, stress tolerance, and drug response research
RNA processing and cell cycle regulation	SRPK	Serine/arginine protein kinases	RNA splicing, post-transcriptional regulation, cell cycle, and tumor proliferation research
Kinase screening research	STK33	Serine/threonine kinase 33	Tumor cell dependency, kinase functional screening, and proliferation regulation research
Kinase screening research	MARK	Microtubule affinity-regulating kinases	Microtubule regulation, cell polarity, cell cycle progression, and stress response research
Kinase screening research	TOPK	T-LAK cell-originated protein kinase	Mitosis, DNA damage tolerance, tumor proliferation, and therapeutic sensitivity research
RNA/DNA-binding regulation research	YB-1	Y-box binding protein 1	DNA repair, transcriptional/translational regulation, drug tolerance, and tumor progression research

Table 6 Representative Products for Cell Cycle and DNA Damage Research

Research Module	Representative Product	CAS No.	Mechanism or Detection Positioning	Applicable Research
DNA crosslink damage induction	Cisplatin	15663-27-1	Forms intra- and interstrand DNA crosslinks and activates DDR	DNA crosslink damage, chemotherapy mechanism, and DDR activation research
DNA alkylation damage induction	Temozolomide	85622-93-1	DNA alkylating agent that induces replication-associated damage	Alkylation damage, MMR-related drug sensitivity, and tumor cell efficacy evaluation
DNA double-strand break induction	Etoposide	33419-42-0	Topoisomerase II inhibitor that induces DSBs	γH2AX, 53BP1 foci, DSB repair, and drug sensitivity research
DNA replication stress induction	Hydroxyurea	127-07-1	Inhibits ribonucleotide reductase and reduces dNTP supply	Replication fork stalling, ATR-Chk1 activation, and S-phase arrest research
DNA synthesis detection	EdU	61135-33-9	Incorporates into newly synthesized DNA to detect S-phase cells	DNA replication, S-phase fraction, and cell proliferation detection
DNA synthesis detection	BrdU	59-14-3	Incorporates into replicating DNA to label DNA synthesis	Cell cycle analysis, S-phase detection, and proliferation evaluation
DNA content staining	Propidium iodide	25535-16-4	DNA fluorescent dye for flow cytometric cell cycle analysis	PI flow cytometry, G1/S/G2-M distribution, and DNA content detection
Nuclear DNA staining	DAPI	28718-90-3	DNA-binding fluorescent dye	Nuclear staining, chromosome morphology, and immunofluorescence co-staining
ATM pathway inhibition	KU-55933	587871-26-9	ATM inhibitor	DSB response, p-ATM/p-Chk2 signaling, and radiosensitization research
ATR pathway inhibition	VE-821	1232410-49-9	ATR inhibitor	Replication stress, ATR-Chk1 signaling, and checkpoint dependency research
Chk1/Chk2 checkpoint inhibition	AZD7762	860352-01-8	Chk1/Chk2 inhibitor	G2/M checkpoint, DNA damage arrest, and combination efficacy research
Wee1 checkpoint inhibition	Adavosertib	955365-80-7	Wee1 inhibitor that releases Cdk1 inhibition	G2/M checkpoint abrogation, Cdk1 Tyr15, and replication stress dependency research
DNA-PK pathway inhibition	NU7441	503468-95-9	DNA-PK inhibitor	NHEJ repair, DSB repair, and radiosensitization research
PARP repair pathway inhibition	Olaparib	763113-22-0	PARP inhibitor	BER, HR deficiency, and BRCA-related synthetic lethality research
CDK4/6-mediated cell cycle arrest	Palbociclib	571190-30-2	CDK4/6 inhibitor	pRB-E2F axis, G1/S arrest, and tumor proliferation inhibition research
Broad-spectrum CDK inhibition	Roscovitine	186692-46-6	CDK inhibitor	CDK activity, cell cycle progression, and proliferation inhibition research
PLK1 mitotic regulation	BI 2536	755038-02-9	PLK1 inhibitor	G2/M transition, spindle formation, and mitotic arrest research
Aurora kinase inhibition	Alisertib	1028486-01-2	Aurora A inhibitor	Centrosome maturation, chromosome alignment, and M-phase abnormality research
Microtubule dynamics intervention	Nocodazole	31430-18-9	Microtubule-destabilizing agent	M-phase synchronization, spindle disruption, and cell cycle arrest research
Microtubule stabilization intervention	Paclitaxel	33069-62-4	Microtubule-stabilizing agent	Mitotic arrest, spindle abnormality, and anti-mitotic efficacy research
Topoisomerase I inhibition	Camptothecin	7689-03-4	TOP1 inhibitor	Replication-transcription conflict, DNA breaks, and topological stress research
HDAC epigenetic regulation	Trichostatin A	58880-19-6	HDAC inhibitor	Chromatin relaxation, DNA repair regulation, and cell cycle arrest research
Sirtuin/metabolic stress regulation	Nicotinamide	98-92-0	NAD⁺-related deacetylation regulatory molecule	Sirtuin pathway, DNA repair, metabolic stress, and senescence research

The cell cycle and DNA damage response jointly determine whether cells can complete proliferation while maintaining genome stability. In research, cell cycle phase, checkpoint activation, damage type, repair capacity, and cell fate should be analyzed together, and mechanistic conclusions should not be drawn from a single phosphorylation marker or change in cell viability alone.

References

[1] Duronio RJ, et al. Cold Spring Harb Perspect Biol. 2013 Mar;5(3):a008904.

[2] Liu W, et al. Mol Cancer. 2017 Mar 14;16(1):60.

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