Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.     

RecA acts in trans to allow replication of damaged DNA by DNA polymerase V

The DNA polymerase V (pol V) and RecA proteins are essential components of a mutagenic translesion synthesis pathway in Escherichia coli designed to cope with DNA damage. Previously, it has been assumed that RecA binds to the DNA template strand being copied. Here we show, however, that pol-V-catalysed translesion synthesis, in the presence or absence of the beta-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble or RecA protomers bind on separate single-stranded (ss)DNA molecules in trans. A 3'-proximal RecA filament end on trans DNA is essential for stimulation; however, synthesis is strengthened by further pol V-RecA interactions occurring elsewhere along a trans nucleoprotein filament. We suggest that trans-stimulation of pol V by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis despite the absolute requirement of RecA for SOS mutagenesis.     

Regulation of p53 activity through lysine methylation

p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase.     

Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA

The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. Here we show that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant.     

MDC1 is required for the intra-S-phase DNA damage checkpoint

MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.     

MDC1 is a mediator of the mammalian DNA damage checkpoint

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal.     

Delayed ageing through damage protection by the Arf/p53 pathway

The tumour-suppressor pathway formed by the alternative reading frame protein of the Cdkn2a locus (Arf) and by p53 (also called Trp53) plays a central part in the detection and elimination of cellular damage, and this constitutes the basis of its potent cancer protection activity. Similar to cancer, ageing also results from the accumulation of damage and, therefore, we have reasoned that Arf/p53 could have anti-ageing activity by alleviating the load of age-associated damage. Here we show that genetically manipulated mice with increased, but otherwise normally regulated, levels of Arf and p53 present strong cancer resistance and have decreased levels of ageing-associated damage. These observations extend the protective role of Arf/p53 to ageing, revealing a previously unknown anti-ageing mechanism and providing a rationale for the co-evolution of cancer resistance and longevity.     

Recognition of DNA damage by the Rad4 nucleotide excision repair protein

Mutations in the nucleotide excision repair (NER) pathway can cause the xeroderma pigmentosum skin cancer predisposition syndrome. NER lesions are limited to one DNA strand, but otherwise they are chemically and structurally diverse, being caused by a wide variety of genotoxic chemicals and ultraviolet radiation. The xeroderma pigmentosum C (XPC) protein has a central role in initiating global-genome NER by recognizing the lesion and recruiting downstream factors. Here we present the crystal structure of the yeast XPC orthologue Rad4 bound to DNA containing a cyclobutane pyrimidine dimer (CPD) lesion. The structure shows that Rad4 inserts a beta-hairpin through the DNA duplex, causing the two damaged base pairs to flip out of the double helix. The expelled nucleotides of the undamaged strand are recognized by Rad4, whereas the two CPD-linked nucleotides become disordered. These findings indicate that the lesions recognized by Rad4/XPC thermodynamically destabilize the Watson-Crick double helix in a manner that facilitates the flipping-out of two base pairs.     

Binding of double-strand breaks in DNA by human Rad52 protein

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.     

彗星电泳检测细胞DNA损伤应用新进展

彗星电泳是近年发展起来的一种测定单个细胞DNA损伤的方法 ,因其操作简单、快速及灵敏等特点 ,应用范围十分广泛 .着重介绍了这一技术的发展、图形分析及应用进展     

Regulation of cell cycle progression and gene expression by H2A deubiquitination

Post-translational histone modifications have important regulatory roles in chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on histone H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X-chromosome inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. Here we report the identification and functional characterization of the major deubiquitinase for histone H2A, Ubp-M (also called USP16). Ubp-M prefers nucleosomal substrates in vitro, and specifically deubiquitinates histone H2A but not H2B in vitro and in vivo. Notably, knockdown of Ubp-M in HeLa cells results in slow cell growth rates owing to defects in the mitotic phase of the cell cycle. Further studies reveal that H2A deubiquitination by Ubp-M is a prerequisite for subsequent phosphorylation of Ser 10 of H3 and chromosome segregation when cells enter mitosis. Furthermore, we demonstrate that Ubp-M regulates Hox gene expression through H2A deubiquitination and that blocking the function of Ubp-M results in defective posterior development in Xenopus laevis. This study identifies the major deubiquitinase for histone H2A and demonstrates that H2A deubiquitination is critically involved in cell cycle progression and gene expression.     

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.