7.7: DNA Damage can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at the right time for damage response in a tightly regulated cell cycle.

Replication stress caused by damaged DNA initiates a carefully choreographed pathway of proteins that respond to the specific type of damage with an appropriate repair mechanism. For example, ionizing radiation that can cause double-stranded breaks in DNA activates ATM protein that sets in motion a chain of molecular interactions that involve repair mechanisms such as Non-homologous End Joining, Homologous Repair, and Nucleotide Excision Repair pathway. Kinases like ATM and ATR respond to replication blocks in two distinct processes that operate on different timescales: (i) relatively fast post-translational modifications like phosphorylation of downstream kinases ultimately leading to the inhibition of the cell cycle phosphatase CDC25 required for CDK activation (ii) slower transcriptional regulations, the most well-studied of which, is the role of p53.

p53 is a transcription factor that can regulate the expression of proteins that play critical roles in cell cycle arrest, apoptosis, or senescence. In healthy cells, p53 is maintained in low concentrations. Upon detecting double-strand breaks, ATM activates p53 by phosphorylation. This results in the expression of the CDK inhibitor p21 and the pro-apoptotic BAX and PUMA proteins. p21 arrests cell cycle by inhibiting cyclin–CDK complexes that phosphorylate proteins mediating G1 to S phase transition. Hence, p53 is critical to the G1/S checkpoint mechanism. In cells where p53 is mutated or absent, cell division can no longer be regulated, and such an uncontrolled cell division results in malignant tumors. Additionally, p53 can directly activate repair pathways such as NER via the regulation of factors that mediate Nucleotide Excision Repair and induce dNTP synthesis.

At various checkpoints during the cell cycle, multiple enzymes probe the DNA for damage. To maintain the integrity of the genome, only intact, undamaged DNA is allowed to pass through this cycle, and on to the next generation. If DNA damage is detected, the cell cycle pauses until this is repaired.

During DNA replication in the S phase, helicase unwinds the DNA, and DNA polymerase synthesizes a new strand from the template - creating a Y-shaped structure called a replication fork. Damaged DNA stalls the replication fork, causing it to become unstable, and the helicase and polymerase to uncouple from the DNA.

To prevent the damaged single-stranded DNA from reannealing, replication protein A or RPA, coats the single-stranded DNA at the stalled replication fork. This complex is then detected by the ATR protein, also known as ataxia telangiectasia or Rad-3 related protein.

If the damaged DNA is not a single mutation but a full double strand break, a protein complex called MRN is recruited at the site, which bridges the two damaged ends of the DNA and provides a platform for binding of the ataxia-telangiectasia mutated, or ATM protein.

Both ATM and ATR are kinases, which means they catalyze the transfer of phosphate groups from phosphate-donating molecules such as NTPs, to specific substrates.

ATR and ATM phosphorylate the downstream kinases Chk1 and Chk2, respectively. Chk1 and Chk2 phosphorylate CDC25, which prevents it from accepting further phosphates from CDK1. CDK1 is the regulator of the cell cycle, and as long as it remains inactive, this prevents the cell from progressing to the S phase.

Another target of ATR and ATM phosphorylation is the transcription activator protein p53. Phosphorylated p53 can directly bind to DNA, which stimulates another gene to produce a protein called p21. p21 inhibits the cell division-stimulating protein cdk2, preventing the cell from progressing to the next stage of cell division.