Spatiotemporal dynamics of regulatory protein recruitment at DNA damage sites

Mammalian cells are constantly threatened by multiple types of DNA lesions arising from various sources like irradiation, environmental agents, replication errors or by-products of the normal cellular metabolism. If not readily detected and repaired these lesions can lead to cell death or to the transformation of cells giving rise to life-threatening diseases like cancer. Multiple specialized repair pathways have evolved to preserve the genetic integrity of a cell. The increasing number of DNA damage sensors, checkpoint regulators, and repair factors identified in the numerous interconnected repair pathways raises the question of how DNA repair is coordinated. In the last decade, various methods have been developed that allow the induction of DNA lesions and subsequent real-time analysis of repair factor assembly at DNA repair sites in living cells. This combination of biophysical and molecular cell biology methods has yielded interesting new insights into the order and kinetics of protein recruitment and identified regulatory sequences and selective loading platforms for the efficient restoration of the genetic and epigenetic integrity of mammalian cells.     

Spatiotemporal recruitment of human DNA polymerase delta to sites of UV damage

Human DNA polymerase δ (Pol δ) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol δ4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol δ4 to Pol δ3, a trimeric form lacking the p12 subunit. We provide the first analysis of the time-dependent recruitment of the individual Pol δ subunits to sites of DNA damage produced by UV irradiation through 5 μm polycarbonate filters by immunofluorescence microscopy and laser scanning cytometry (LSC). Quantitative analysis demonstrates that the recruitments of the three large subunits was near complete by 2 h and did not change significantly up to 4 h after UV exposure. However, the recruitment of p12 was incomplete even at 4 h, with about 70% of the Pol δ lacking the p12 subunit. ChIP analysis of Pol δ after global UV irradiation further demonstrates that only p125, p50 and p68 were present. Thus, Pol δ3 is the predominant form of Pol δ at sites of UV damage as a result of p12 degradation. Using LSC, we have further confirmed that Pol δ was recruited to CPD damage sites in all phases of the cell cycle. Collectively, our results show that Pol δ at the DNA damage site is the Pol δ trimer lacking p12 regardless of the cell cycle phase.     

RAD51调控REV1参与DNA双链断裂修复

REV1是跨损伤聚合酶Y家族的重要成员之一,它不仅作为支架蛋白介导Y家族聚合酶招募至损伤位点完成跨损伤DNA合成(translesion DNA synthesis, TLS),还可利用自身的dCMP转移酶活性在一些损伤位点对侧整合dCMP参与TLS。此外,REV1也被报导参与调控同源重组修复。为进一步探讨REV1互作蛋白RAD51和RAD51C在其参与的同源重组修复通路中的调控作用,本研究采用脉冲氮激光微辐射实验,发现RAD51可调控REV1到双链断裂位点的募集。同时,免疫荧光实验结果证明REV1也反过来影响RAD51应答CPT损伤。然而敲低RAD51C并不影响REV1到DNA双链断裂位点的招募。结果表明,REV1和RAD51在HR通路中存在彼此相互调控的关系。     

Active DNA damage eviction by HLTF stimulates nucleotide excision repair

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Role for RIF1-interacting partner DDX1 in BLM recruitment to DNA double-strand breaks

Human Rap1-interacting factor 1 (RIF1) is an important player in the repair of DNA double strand breaks (DSBs). RIF1 acts downstream of 53BP1, with well-documented roles in class switch recombination in B-cells and inhibition of end resection initiation in BRCA1-defective cells. Here, we report that DEAD Box 1 (DDX1), a RNA helicase also implicated in DSB repair, interacts with RIF1, with co-localization of DDX1 and RIF1 observed throughout interphase. Recruitment of DDX1 to DSBs is dependent on RIF1, with RIF1 depletion abolishing DDX1-mediated facilitation of homologous recombination at DSBs. As previously demonstrated for RIF1, DDX1 is also required for chromatin loading of Bloom syndrome helicase (BLM) to ionizing radiation-induced DSBs, a RIF1-related activity that is independent of 53BP1. We show that DDX1 and RIF1 have different nucleic acid requirements for accumulation at DSBs, with RNA-DNA hybrids required for DDX1 accrual at DSBs, and single-strand RNA required for accumulation of RIF1 at these sites. Our data suggest both convergent and divergent roles for DDX1 and RIF1 in DSB repair, and may help explain why RIF1 depletion does not fully mimic 53BP1 ablation in the restoration of homologous recombination defects in BRCA1-deficient cells.     

Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases

Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2-7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members.     

Molecular insights into DNA polymerase deterrents for ribonucleotide insertion

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase β discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn²⁺ for Mg²⁺ decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2′ of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.     

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.     

Structural insights into the cryptic DNA-dependent ATPase activity of UvrB

The UvrABC pathway is a ubiquitously occurring mechanism targeted towards the repair of bulky base damage. Key to this process is UvrB, a DNA-dependent limited helicase that acts as a lesion recognition element whilst part of a tracking complex involving UvrA, and as a DNA-binding platform required for the presentation of damage to UvrC for subsequent processing. We have been able to determine the structure of a ternary complex involving UvrB* (a C-terminal truncation of full-length UvrB), a polythymine trinucleotide and ADP. This structure has highlighted the roles of key conserved residues in DNA binding distinct from those of the beta-hairpin, where most of the attention in previous studies has been focussed. We are also the first to report the structural basis underlying conformational re-modelling of the beta-hairpin that is absolutely required for DNA binding and how this event results in an ATPase primed for catalysis. Our data provide the first insights at the molecular level into the transformation of UvrB into an active helicase.     

The repair of DNA damage: Recent developments and new insights

This brief review presents the salient features of new developments in the enzymatic repair of base damage to DNA. DNA glycosylases and apurinic/ apyrimidinic (AP) endonucleases are reviewed and evidence is presented that in at least two prokaryote systems incision of UV-irradiated DNA occurs by the sequential action of these two classes of enzymes. In contradistinction, the uvrA, uvrB, and uvrC gene products of E coli appear to function as a multiprotein complex that catalyzes hydrolysis of phosphodiester bonds in damaged DNA directly. The inducible rapid repair of O⁶- methylguanine in E coli is also reviewed.     

Archaeal Hel308 domain V couples DNA binding to ATP hydrolysis and positions DNA for unwinding over the helicase ratchet

Hel308 and PolQ are paralogues with roles promoting genome stability in archaea and higher eukaryotes. The context in which they act is not clear, although Hel308 helicase from archaea may interact with abnormal replication forks. The atomic structure of archaeal Hel308 from Archaeoglobus fulgidus in complex with DNA was recently reported and has given insights into the mechanisms of superfamily-2 helicases generally. An intriguing aspect of the structure was the positioning of a C-terminal domain V relative to single-stranded DNA and to the helicase ratchet domain IV. We have mutagenised a triplet of arginine residues in domain V of archaeal Hel308 to assess the effects on DNA binding, unwinding, and ATPase activities. Our observations can now be interpreted in light of the atomic structure. We describe crucial roles for domain V as a brake on ATP hydrolysis by coupling it to binding single-stranded DNA and in positioning DNA relative to the helicase ratchet domain IV for efficient unwinding of forked DNA.     

SUMOylation and PARylation cooperate to recruit and stabilize SLX4 at DNA damage sites

SUMOylation plays important roles in the DNA damage response. However, whether it is important for interstrand crosslink repair remains unknown. We report that the SLX4 nuclease scaffold protein is regulated by SUMOylation. We have identified three SUMO interaction motifs (SIMs) in SLX4, mutating all of which abrogated the binding of SLX4 to SUMO-2 and covalent SLX4 SUMOylation. An SLX4 mutant lacking functional SIMs is not recruited to PML nuclear bodies nor stabilized at laser-induced DNA damage sites. Additionally, we elucidated a novel role for PARylation in the recruitment of SLX4 to sites of DNA damage. Combined, our results uncover how SLX4 is regulated by post-translational modifications.     

DNA修复的分子机制

ＤＮＡ是遗传信息的载体,需要有极高的保真度,这不仅有赖于完善的复制体系,而且还需要有能纠正已存在错误的修复系统。对于不同的ＤＮＡ损伤,生物体内存在许多不同的修复系统。本文介绍三种主要修复系统即核苷酸切割修复,错配修复及转录偶联修复的分子机制,深入研究ＤＮＡ修复作用对了解某些癌症成因及细胞衰老等过程有重要意义 。     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC → AT transitions and GC → TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to asses the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.