Opposite roles of Rad5 in DNA damage tolerance: playing in both error-free and mutagenic lesion bypass

DNA Damage Tolerance (DDT) funcPons to bypass replicaPon-blocking lesions and is divided into two disPnct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is an important player in these processes. Indeed, Saccharomyces cerevisiae Rad5 is a large mulPfuncPonal protein that contains three well defined domains: a RING domain that promotes PCNA polyubiquiPnaPon and a ssDNA-dependent ATPase/helicase domain, that are both conserved in Rad5 human ortholog HLTF. Yeast Rad5 also contains a Rev1-binding domain. In this study we used domain-specific mutants to address the contribuPon of each of the Rad5 funcPons to lesion tolerance. Using an assay based on the inserPon of a single lesion into a defined locus in the genome of a living yeast cell, we demonstrate that Rad5 plays opposite roles in lesion tolerance: i) Rad5 favors error-free lesion bypass by acPvaPng template switching through polyubiquiPnaPon of PCNA; ii) Rad5 is also required for TLS by recruiPng the TLS polymerase Rev1. We also show that the helicase acPvity does not play any role in lesion tolerance/

Enzymatic Switching Between Archaeal DNA Polymerases Facilitates Abasic Site Bypass

Abasic sites are among the most abundant DNA lesions encountered by cells. Their replication requires actions of specialized DNA polymerases. Herein, two archaeal specialized DNA polymerases were examined for their capability to perform translesion DNA synthesis (TLS) on the lesion, including Sulfolobuss islandicus Dpo2 of B-family, and Dpo4 of Y-family. We found neither Dpo2 nor Dpo4 is efficient to complete abasic sites bypass alone, but their sequential actions promote lesion bypass. Enzyme kinetics studies further revealed that the Dpo4’s activity is significantly inhibited at +1 to +3 site past the lesion, at which Dpo2 efficiently extends the primer termini. Furthermore, their activities are inhibited upon synthesis of 5–6 nt TLS patches. Once handed over to Dpo1, these substrates basically inactivate its exonuclease, enabling the transition from proofreading to polymerization of the replicase. Collectively, by functioning as an “extender” to catalyze further DNA synthesis past the lesion, Dpo2 bridges the activity gap between Dpo4 and Dpo1 in the archaeal TLS process, thus achieving more efficient lesion bypass.

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance

The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.

Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

The HLTF–PARP1 interaction in the progression and stability of damaged replication forks caused by methyl methanesulfonate

Human HLTF participates in the lesion-bypass mechanism through the fork reversal structure, known as template switching of post-replication repair. However, the mechanism by which HLTF promotes the replication progression and fork stability of damaged forks remains unclear. Here, we identify a novel protein–protein interaction between HLTF and PARP1. The depletion of HLTF and PARP1 increases chromosome breaks, further reduces the length of replication tracks, and concomitantly increases the number of stalled forks after methyl methanesulfonate treatment according to a DNA fiber analysis. The progression of replication also depends on BARD1 in the presence of MMS treatment. By combining 5-ethynyl-2′-deoxyuridine with a proximity ligation assay, we revealed that the HLTF, PARP1, and BRCA1/BARD1/RAD51 proteins were initially recruited to damaged forks. However, prolonged stalling of damaged forks results in fork collapse. HLTF and PCNA dissociate from the collapsed forks, with increased accumulation of PARP1 and BRCA1/BARD1/RAD51 at the collapsed forks. Our results reveal that HLTF together with PARP1 and BARD1 participates in the stabilization of damaged forks, and the PARP1–BARD1 interaction is further involved in the repair of collapse forks.