Strand dependent bypass of DNA lesions during fork reversal by ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF.

During DNA replication, the replisome encounters obstacles including DNA lesions, transcription-replication conflicts, and other sources of replication stress. These obstacles must be efficiently overcome to complete DNA synthesis and minimize genome instability. One pathway to tolerate replication stress is replication fork reversal, in which parental template DNA strands are reannealed and a nascent-nascent DNA duplex is formed. Several enzymes promote replication fork reversal, including the ATP-dependent translocases SMARCAL1, ZRANB3, and HLTF. How these enzymes translocate on DNA that contains fork-stalling lesions is unknown. Here, we examined the abilities of SMARCAL1, ZRANB3, and HLTF to tolerate various lesions on leading or lagging template strands. We demonstrate that SMARCAL1 and ZRANB3 are selectively inhibited by lesions on the leading template strand, whereas HLTF is insensitive to bulky lesions on either strand. These results suggest that SMARCAL1 and ZRANB3 contact the leading strand during fork reversal and therefore are more sensitive to inhibition by bulky lesions on this strand. In contrast, HLTF DNA translocation is inherently insensitive to DNA lesions. These biochemical differences between the fork reversal enzymes provide insights into their mechanism of DNA remodeling and suggest they may act in lesion-specific contexts.

Mechanisms and regulation of replication fork reversal.

DNA replication is remarkably accurate with estimates of only a handful of mutations per human genome per cell division cycle. Replication stress caused by DNA lesions, transcription-replication conflicts, and other obstacles to the replication machinery must be efficiently overcome in ways that minimize errors and maximize completion of DNA synthesis. Replication fork reversal is one mechanism that helps cells tolerate replication stress. This process involves reannealing of parental template DNA strands and generation of a nascent-nascent DNA duplex. While fork reversal may be beneficial by facilitating DNA repair or template switching, it must be confined to the appropriate contexts to preserve genome stability. Many enzymes have been implicated in this process including ATP-dependent DNA translocases like SMARCAL1, ZRANB3, HLTF, and the helicase FBH1. In addition, the RAD51 recombinase is required. Many additional factors and regulatory activities also act to ensure reversal is beneficial instead of yielding undesirable outcomes. Finally, reversed forks must also be stabilized and often need to be restarted to complete DNA synthesis. Disruption or deregulation of fork reversal causes a variety of human diseases. In this review we will describe the latest models for reversal and key mechanisms of regulation.

RTEL1 helicase counteracts RAD51-mediated homologous recombination and fork reversal to safeguard replicating genomes.

Homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSBs), replication stress responses, and genome maintenance. However, unregulated HR during replication can impair genome duplication and compromise genome stability. The mechanisms underlying HR regulation during DNA replication are obscure. Here, we find that RTEL1 helicase, RAD51, and RAD51 paralogs are enriched at stalled replication sites. The absence of RTEL1 leads to an increase in the RAD51-mediated HR and fork reversal during replication and affects genome-wide replication, which can be rescued by co-depleting RAD51 and RAD51 paralogs. Interestingly, co-depletion of fork remodelers such as SMARCAL1/ZRANB3/HLTF/FBH1 and expression of HR-defective RAD51 mutants also rescues replication defects in RTEL1-deficient cells. The anti-recombinase function of RTEL1 during replication depends on its interaction with PCNA and helicase activity. Together, our data identify the role of RTEL1 helicase in restricting RAD51-mediated fork reversal and HR activity to facilitate error-free genome duplication.

FANCM branchpoint translocase: Master of traverse, reverse and adverse DNA repair.

FANCM is a multifunctional DNA repair enzyme that acts as a sensor and coordinator of replication stress responses, especially interstrand crosslink (ICL) repair mediated by the Fanconi anaemia (FA) pathway. Its specialised ability to bind and remodel branched DNA structures enables diverse genome maintenance activities. Through ATP-powered "branchpoint translocation", FANCM can promote fork reversal, facilitate replication traverse of ICLs, resolve deleterious R-loop structures, and restrain recombination. These remodelling functions also support a role as sensor of perturbed replication, eliciting checkpoint signalling and recruitment of downstream repair factors like the Fanconi anaemia FANCI:FANCD2 complex. Accordingly, FANCM deficiency causes chromosome fragility and cancer susceptibility. Other recent advances link FANCM to roles in gene editing efficiency and meiotic recombination, along with emerging synthetic lethal relationships, and targeting opportunities in ALT-positive cancers. Here we review key properties of FANCM's biochemical activities, with a particular focus on branchpoint translocation as a distinguishing characteristic.

Pold4 subunit of replicative polymerase δ promotes fork slowing at broken templates.

Single-strand breaks (SSBs) are the most frequent type of lesion, and replication across such lesions leads to double-strand breaks (DSBs). DSBs that arise during replication are repaired by homologous recombination (HR) and are suppressed by fork reversal. Poly[ADP-ribose] polymerase I (PARP1) and the proofreading exonuclease activity of replicative polymerase ε (Polε) are required for fork reversal when leading strand replication encounters SSBs. However, the mechanism underlying fork reversal at the SSB during lagging-strand replication remains elusive. We here demonstrate that the Pold4 subunit of replicative polymerase δ (Polδ) plays a role in promoting fork reversal during lagging strand replication on a broken template. POLD4-/- cells exhibited heightened sensitivity to camptothecin (CPT) but not to other DNA-damaging agents compared to wild-type cells. This selective CPT sensitivity in POLD4-/- cells suggests that Pold4 suppresses DSBs during replication, as CPT induces significant SSBs during replication, which subsequently lead to DSBs. To explore the functional interactions among Pold4, Polε exonuclease, and PARP1 in DSB suppression, we generated PARP1-/-, POLD4-/-, Polε exonuclease-deficient POLE1exo-/-, PARP1-/-/POLD4-/-, and POLD4-/-/POLE1exo-/- cells. These epistasis analyses showed that Pold4 is involved in the PARP1-Polε exonuclease-mediated fork reversal following CPT treatment. These results suggest that Pold4 aids in fork reversal when lagging strand replication stalls on a broken template. In conclusion, the Pold4 subunit of Polδ has roles in the PARP1-Polε exonuclease-mediated fork reversal, contributing to the suppression of DSBs.

Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection.

Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.

Processing of stalled replication forks in Bacillus subtilis.

Accurate DNA replication and transcription elongation are crucial for preventing the accumulation of unreplicated DNA and genomic instability. Cells have evolved multiple mechanisms to deal with impaired replication fork progression, challenged by both intrinsic and extrinsic impediments. The bacterium Bacillus subtilis, which adopts multiple forms of differentiation and development, serves as an excellent model system for studying the pathways required to cope with replication stress to preserve genomic stability. This review focuses on the genetics, single molecule choreography, and biochemical properties of the proteins that act to circumvent the replicative arrest allowing the resumption of DNA synthesis. The RecA recombinase, its mediators (RecO, RecR, and RadA/Sms) and modulators (RecF, RecX, RarA, RecU, RecD2, and PcrA), repair licensing (DisA), fork remodelers (RuvAB, RecG, RecD2, RadA/Sms, and PriA), Holliday junction resolvase (RecU), nucleases (RnhC and DinG), and translesion synthesis DNA polymerases (PolY1 and PolY2) are key functions required to overcome a replication stress, provided that the fork does not collapse.

Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability.

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.

Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand.

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5' overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.

Rad5 HIRAN domain: Structural insights into its interaction with ssDNA through molecular modeling approaches.

The Rad5 protein is an SWI/SNF family ubiquitin ligase that contains an N-terminal HIRAN domain and a RING C3HC4 motif. The HIRAN domain is critical for recognition of the stalled replication fork during the replication process and acts as a sensor to initiate the damaged DNA checkpoint. It is a conserved domain widely distributed in eukaryotic organisms and is present in several DNA-binding proteins from all kingdoms. Here we showed that distant species have important differences in key residues that affect affinity for ssDNA. Based on these findings, we hypothesized that different HIRAN domains might affect fork reversal and translesion synthesis through different metabolic processes. To address this question, we predicted the tertiary structure of both yeast and human HIRAN domains using molecular modeling. Structural dynamics experiments showed that the yeast HIRAN domain exhibited higher structural denaturation than its human homolog, although both domains became stable in the presence of ssDNA. Analysis of atomic contacts revealed that a greater number of interactions between the ssDNA nucleotides and the Rad5 domain are electrostatic. Taken together, these results provide new insights into the molecular mechanism of the HIRAN domain of Rad5 and may guide us to further elucidate differences in the ancient eukaryotes HIRAN sequences and their DNA affinity.Communicated by Ramaswamy H. Sarma.

DNA-PKcs promotes fork reversal and chemoresistance.

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

Role of Cockayne Syndrome Group B Protein in Replication Stress: Implications for Cancer Therapy.

A variety of endogenous and exogenous insults are capable of impeding replication fork progression, leading to replication stress. Several SNF2 fork remodelers have been shown to play critical roles in resolving this replication stress, utilizing different pathways dependent upon the nature of the DNA lesion, location on the DNA, and the stage of the cell cycle, to complete DNA replication in a manner preserving genetic integrity. Under certain conditions, however, the attempted repair may lead to additional genetic instability. Cockayne syndrome group B (CSB) protein, a SNF2 chromatin remodeler best known for its role in transcription-coupled nucleotide excision repair, has recently been shown to catalyze fork reversal, a pathway that can provide stability of stalled forks and allow resumption of DNA synthesis without chromosome breakage. Prolonged stalling of replication forks may collapse to give rise to DNA double-strand breaks, which are preferentially repaired by homology-directed recombination. CSB plays a role in repairing collapsed forks by promoting break-induced replication in S phase and early mitosis. In this review, we discuss roles of CSB in regulating the sources of replication stress, replication stress response, as well as the implications of CSB for cancer therapy.

The Biochemical Mechanism of Fork Regression in Prokaryotes and Eukaryotes-A Single Molecule Comparison.

The rescue of stalled DNA replication forks is essential for cell viability. Impeded but still intact forks can be rescued by atypical DNA helicases in a reaction known as fork regression. This reaction has been studied at the single-molecule level using the Escherichia coli DNA helicase RecG and, separately, using the eukaryotic SMARCAL1 enzyme. Both nanomachines possess the necessary activities to regress forks: they simultaneously couple DNA unwinding to duplex rewinding and the displacement of bound proteins. Furthermore, they can regress a fork into a Holliday junction structure, the central intermediate of many fork regression models. However, there are key differences between these two enzymes. RecG is monomeric and unidirectional, catalyzing an efficient and processive fork regression reaction and, in the process, generating a significant amount of force that is used to displace the tightly-bound E. coli SSB protein. In contrast, the inefficient SMARCAL1 is not unidirectional, displays limited processivity, and likely uses fork rewinding to facilitate RPA displacement. Like many other eukaryotic enzymes, SMARCAL1 may require additional factors and/or post-translational modifications to enhance its catalytic activity, whereas RecG can drive fork regression on its own.

MDM2 binds and ubiquitinates PARP1 to enhance DNA replication fork progression.

The MDM2 oncoprotein antagonizes the tumor suppressor p53 by physical interaction and ubiquitination. However, it also sustains the progression of DNA replication forks, even in the absence of functional p53. Here, we show that MDM2 binds, inhibits, ubiquitinates, and destabilizes poly(ADP-ribose) polymerase 1 (PARP1). When cellular MDM2 levels are increased, this leads to accelerated progression of DNA replication forks, much like pharmacological inhibition of PARP1. Conversely, overexpressed PARP1 restores normal fork progression despite elevated MDM2. Strikingly, MDM2 profoundly reduces the frequency of fork reversal, revealed as four-way junctions through electron microscopy. Depletion of RECQ1 or the primase/polymerase (PRIMPOL) reverses the MDM2-mediated acceleration of the nascent DNA elongation rate. MDM2 also increases the occurrence of micronuclei, and it exacerbates camptothecin-induced cell death. In conclusion, high MDM2 levels phenocopy PARP inhibition in modulation of fork restart, representing a potential vulnerability of cancer cells.

TT-pocket/HIRAN: binding to 3'-terminus of DNA for recognition and processing of stalled replication forks.

Stalled replication forks need to be swiftly detected and protected from collapse and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3$^\prime$-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3'-terminus of DNA and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3'-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3'-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.

Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability.

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.

Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling.

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

Genomic mapping of DNA-repair reaction intermediates in living cells with engineered DNA structure-trap proteins.

Diverse DNA structures occur as reaction intermediates in various DNA-damage and -repair mechanisms, most of which results from replication stress. We harness the power of proteins evolutionarily optimized to bind and "trap" specific DNA reaction-intermediate structures, to quantify the structures, and discern the mechanisms of their occurrence in cells. The engineered proteins also allow genomic mapping of sites at which specific DNA structures occur preferentially, using a structure-trapping protein and ChIP-seq- or Cut-and-Tag-like methods. Genome-wide identification of sites with recurrent DNA-damage intermediates has illuminated mechanisms implicated in genome instability, replication stress, and chromosome fragility. Here, we describe X-seq, for identifying sites of recurrent four-way DNA junctions or Holliday-junctions (HJs). X-seq uses an engineered, catalysis-defective mutant of Escherichia coli RuvC HJ-specific endonuclease, RuvCDefGFP. X-seq signal indicates sites of recombinational DNA repair or replication-fork stalling and reversal. We also describe methods for genomic mapping of 3'-single-stranded DNA ends with SsEND-seq, in E. coli. Both methods allow genomic profiling of DNA-damage and -repair intermediates, which can precede genome instability, and are expected to have many additional applications including in other cells and organisms.

DisA Restrains the Processing and Cleavage of Reversed Replication Forks by the RuvAB-RecU Resolvasome.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.