DNA Lesions IV

Homologous Recombination (HR)

When replication fails catastrophically due to a lesion on the leading-strand template, the DNA being copied — the leading nascent strand — can break or become chewed back, leaving its 3′ end--the end that DNA polymerase epsilon needs to restart--located behind the polymerase, where it is inaccessible to the polymerase.

If the fork simply tries to resume, it can’t: there is no usable 3' end starting point. At this point, the fork reverses, and the two newly made strands pair with one another to form a short double-stranded “arm” behind the fork--the regressed arm.

At this moment the cell makes a crucial decision.The leading nascent strand — the one that was damaged — is gently processed at its broken end, exposing a small region of single-stranded DNA.

RAD51 coats this exposed tail like beads sliding onto a wire.

This RAD51-coated strand now becomes active.It searches for the matching sequence in the sister chromatid, which carries a perfect, undamaged version of the same region of the leading-strand template and its nascent copy.

When the RAD51-coated leading nascent strand finds that matching sequence, it pushes into the intact double helix, briefly opening it and pairing with its complementary bases.This step — strand invasion — gives the damaged strand access to the missing genetic information.

A DNA polymerase then uses the intact sister copy as a guide, extending the damaged leading nascent strand and rebuilding whatever section was lost.Once the missing piece is restored, the strands disengage, the reversed fork is “unfolded,” and replication can restart.

Global Response

In addition to the local response to a lesion initiated by phosphorylated CHK1 (that is, fork stabilization in the form of fork protection and potentially fork reversal) the presence of high levels of phosphorylated CHK1 also contribute to a call for a global, cell-wide response. The cell-wide response takes the form of cell cycle arrest at the intra-S-phase checkpoint. This buys time for the cell to address what now appears to be (based on very high levels of phosphorylated CHK1) a global replication problem--not just a lesion at a small number of sites.

Here's how it works: Recall that the phases of the cell cycle are determined by proteins called cyclin-dependent kinases (CDKs) and their partner proteins, various cyclins roaming around the nucleus. S-phase, specifically, is driven (or "turned on") by CDK2 and cyclins E (earlier S-phase) and A (later S-phase). If the cell wanted to arrest the cell cycle at the intra-S checkpoint because it sensed serious replication stress, then CHK1 would have to suppress CDK2 or these cyclins for phosphorylation.

Intra-S checkpoint. The cell gets a kind of "status report" at different points in the cell cycle called "checkpoints." The Intra-S checkpoint is focused on DNA replication. If there is significant replication stress within the cell as measured by the amount of activated CHK1 in the nucleus, the cell will arrest the cell cycle

In fact, CHK1 suppresses CDK2 rather than the cyclins--but not directly. Here's the story. In its inactive form, CDK2 has two inhibitory phosphates attached to two specific amino acids (Ser76 and Ser124). When CDK2 activity is needed, a phosphatase (a protein that removes phosphates, as opposed to a kinase, which is a protein that adds phosphates) called CDC25A is expressed and removes these two phosphates, maintaining CDK2 activity.

But when a global response to replication stress is justified, a lot of CHK1s phosphorylate a lot of CDC25As at the two serine amino acids. These phosphorylations both suppress the CDC25As' phosphatase activity and target them for rapid degradation. In this high CHK1 environment there won't be many CDC25As to de-phosphorylate (and thus activate) CDK2s. Thus, the CDK2s become re-phosphorylated and inactivated. This leads to cell cycle arrest at the intra-S checkpoint, which buys the cell time to either address whatever problem is hindering replication or take steps to dismantle itself in a process called apoptosis.

Having just introduced the concept of apoptosis, let me say a few words about it. Apoptosis is usually given the moniker "programmed cell death." If a cell senses that DNA replication is in serious trouble and risks producing a highly damaged genome, it initiates a internal process in which it methodically dismantles itself molecular piece by molecular piece, recycling biomolecules that can then be used by other cells. Apoptosis is referred to more evocatively as "programmed cell suicide." Again, the problem with a highly damaged genome is that it can lead a cell down a path towards becoming malignant, or cancerous.

But again, the cell won't arrest S-phase unless it sense replication problems at numerous sites on the genome. It senses this based on the amount of phosphorylated CHK1 in the nucleus. In other words, the amount of phosphorylated CHK1 determines the degree to which CDC25A is phosphorylated and subsequently destroyed. And the extent to which CDC25A is phosphorylated and subsequently destroyed determines the fate of CDK2 and the status of the cell cycle.