Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin

Abstract

All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual's mutation burden and risk of cancer. This review describes radiological, chemical, and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis).

FIGURE 1. DSB formation via attempted base excision repair of closely opposed oxidative lesions

Reactive oxygen species (ROS) can produce oxidized DNA bases, sites of base loss and DNA nicks. The base excision repair (BER) of single lesions (left) begins with the excision of oxidized bases (Ox) by DNA glycosylases, or with the excision of apurinic/apyrimidinic (AP) sites by apurinic endonuclease. In either case, apurinic endonuclease goes on to generate a single base gap that is filled by DNA polymerase β and sealed by DNA ligase IIIα, in complex with XRCC1. Because a single IR particle can produce multiple clustered ROS, IR often generates a cluster of oxidative lesions in DNA. Near-simultaneous BER of closely opposed lesions in such a cluster can generate closely spaced nicked or gapped repair intermediates in opposing DNA strands. These may spontaneously convert to DSBs before BER is complete (right).

FIGURE 2. DSB formation via replication fork collapse

(Left) In the simplest scenario, leading strand synthesis is halted by a single-strand break (SSB), created via exogenous damaging agents or as a repair intermediate (see Figure 1). Collapse of the replication fork converts the SSB into a one-sided DSB. The attempted repair of one-sided DSBs by NHEJ would potentially lead to chromosomal rearrangements or translocations. More commonly, one-sided DSBs may initiate break-induced DNA replication (reviewed in (Malkova and Ira, 2013)). (Right) Several kinds of DNA lesions, including thymine dimers, certain oxidized bases (e.g. thymine glycol), abasic sites, and inter-strand crosslinks (such as those caused by cisplatin) can cause replication forks to stall. If the stalled replication fork regresses, it will partially displace newly synthesized leading and lagging strands, allowing them to anneal, as depicted. The newly synthesized lagging strand may then serve as a template to further extend the leading strand, producing the “chicken foot” intermediate shown. This may resolve if the replication block is removed, allowing replication to restart. Alternatively, because the chicken foot is structurally analogous to a Holliday Junction, it may be cleaved by resolvases, producing, once again, a one-sided DSB.

FIGURE 3. Homology-directed recombination-mediated repair (HRR) via synthesis-dependent strand annealing (SDSA)

The Figure depicts key factors and the sequence of steps in SDSA, a common homology-mediated DSB repair sub-pathway. For clarity and simplicity, other HR-initiated pathways, accessory proteins and replication machinery are not shown, but are described in (Mehta and Haber, 2014; San Filippo et al., 2008) and other HRR-related reviews. Step (A) depicts the initiation of HRR by the MRN complex which, together with the endonuclease Sae2/Ctp1/CtIP resects 5’ DNA ends. This creates 3’ single strand DNA (ssDNA) tails that are bound by the ssDNA binding protein RPA (step (B)). RPA is then replaced by RAD51, which catalyzes a homology search; annealing of the Rad51-ssDNA filament to its homolog displaces the homolog’s normal complement, creating a “D-loop” (step (C)). Using the sister chromatid as a template, the invading strand is able to prime DNA synthesis, thereby extending the D-loop (step (D)). Further extension of the D-loop may enable it to anneal with the second 3’-ssDNA tail, which could then prime DNA synthesis in the opposite direction (not shown). Alternatively, the original, newly-extended invading strand may be displaced, allowing it to anneal with the second 3’-ssDNA tail (step (E)). DNA synthesis primed by the second 3’-ssDNA tail, followed by ligation, would then complete the repair (step (F)).

FIGURE 4. Nucleosomes suppress BER-mediated double strand break formation

(A) depicts the excision of an oxidized base (red hexagon) from a nucleosome by a DNA glycosylase (“Gly”). This excision reaction is relatively high efficient when the lesion is oriented so that it can flip through the major groove (red arrow) without steric hindrance from the histone octamer or nearby DNA, into the active site of the glycosylase, which must be able to bind via the minor groove (white arrow). Provided both these constraints are satisfied, base excision repair can proceed to completion, as depicted in Figure 1 (left). If DNA glycosylases initiate repair of two, closely-spaced lesions on opposing strands at about the same time, subsequent steps in BER will generate single strand break or gapped repair intermediates. If these intermediate are present at the same time they may spontaneously convert into a DSB, as described in Figure 1 (right). However, if the opposing strand lesions are separated by fewer than 3 bps, as depicted in (B), near-simultaneous repair cannot occur, probably because processing of one lesion degrades the binding site needed to initiate repair of the second lesion. This restriction is evident in repair reactions with both DNA and nucleosomal substrates. If the opposing strand lesions are more optimally spaced with respect to one another (e.g. 3 or 7 bp), access to one or both lesions may be hindered by the histone octamer, as depicted in (C). In this case, the more accessible lesion will likely be repaired more rapidly than the opposing strand lesion, where repair can begin only when the lesion is exposed by spontaneous, transient partial unwrapping of DNA from the histone octamer (Maher et al., 2013; Prasad et al., 2007). If the opposing strand lesions are optimally spaced (~4–6 bp), and optimally oriented with respect to the underlying histone octamer, as depicted in (D), near-simultaneous BER may ensue, resulting in a DSB (for additional details, see (Cannan et al., 2014)).