Making Choices: DNA Replication Fork Recovery Mechanisms

Abstract

DNA replication is laden with obstacles that slow, stall, collapse, and break DNA replication forks. At each obstacle, there is a decision to be made whether to bypass the lesion, repair or restart the damaged fork, or to protect stalled forks from further demise. Each "decision" draws upon multitude of proteins participating in various mechanisms that allow repair and restart of replication forks. Specific functions for many of these proteins have been described and an understanding of how they come together in supporting replication forks is starting to emerge. Many questions, however, remain regarding selection of the mechanisms that enable faithful genome duplication and how "normal" intermediates in these mechanisms are sometimes funneled into "rogue" processes that destabilize the genome and lead to cancer, cell death, and emergence of chemotherapeutic resistance. In this review we will discuss molecular mechanisms of DNA damage bypass and replication fork protection and repair. We will specifically focus on the key players that define which mechanism is employed including: PCNA and its control by posttranslational modifications, translesion synthesis DNA polymerases, molecular motors that catalyze reversal of stalled replication forks, proteins that antagonize fork reversal and protect reversed forks from nucleolytic degradation, and the machinery of homologous recombination that helps to reestablish broken forks. We will also discuss risks to genome integrity inherent in each of these mechanisms.

Keywords: BRCA2; DNA replication; HLTF; PCNA; RAD51; RAD52; RPA; SHPRH; SMARCAL1; ZRANB3; genome stability; replication fork protection; replication fork reversal; template switching; translesion synthesis; translesion synthesis DNA polymerases.

Figure 1.. Replication forks encounter numerous impediments to their progression.

On linear eukaryotic chromosomes, DNA replication forks move bidirectionally from replication origins (ori) and can stall or brake due to encounter with protein DNA complexes (PDC), modified nucleobases, difficult to replicate regions, single-strand breaks (SSBs), interstrand DNA crosslinks (ICLs) and ongoing RNA transcription.

Figure 2.. Cells employ multiple mechanisms to process, repair and restart stalled and damaged DNA replication forks.

Nature of the replication stalling event in part defines the mechanism of fork protection and restart. a.) Translesion DNA synthesis (TLS) is triggered by ubiquitylation of PCNA and is carried out by specialized DNA polymerases. b). Poly-ubiquitylation of PCNA promotes template switching (TS). Stalled forks can also recruit fork reversal motors (c.) or enzymes that cleave replication forks (d.). e.) Fork reversal generates so-called “chicken foot” structure whose double strand end requires protection from uncontrolled resection by DSB resection machinery, but which can also serve to repair the fork by homologous recombination (HR; f.). g.) One ended DSB generated by fork cleavage or running into SSB is also processed by the DSB resection machinery allowing the fork to be repaired by HR (h.).

Figure 3.. Structures of damaged DNA templates and incoming dNTPs in the active sites of translesion synthesis polymerases.

a.) DNA polymerase η incorporating dATP opposite the 5’ T of a thymine dimer (173). b.) DNA polymerase iota incorporating dCTP opposite an N²-ethyl-guanine (44). c.) DNA kappa iota incorporating dCTP opposite a benzo(a)pyrene diol epoxide (BPDE)-adducted guanine (174). d.) Rev1 incorporating dCTP opposite a 1,N²-propano-guanine (65).

Figure 4.. Possible mutagenic consequences of template switching.

a.) BIR is prone to template switching, which can yield large insertions and rearrangements when multiple forks are involved. b.) TS between merging forks can result in deletions.

Figure 5.. Replication fork protection.

a.) Fork reversal motors and fork protection proteins are recruited to demised forks by different structural features and proteins already present at the fork. b.) HIRAN domain of HLTF interacts with 3’-OH group of the nascent strand; in the absence of HLTF, forks progression depends on repriming by PRIMPOL. FBH1 and RADX prevent RAD51 binding at replication forks and subsequent fork reversal, while RAD52 gatekeeper function restricts access by the fork reversal motors. c.) Recombination machinery, and especially BRCA2 and RAD51 protect reversed forks from excessive degradation, which can have genome destabilizing consequences.