Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation

Protein modification by ubiquitin is emerging as a signal for various biological processes in eukaryotes, including regulated proteolysis, but also for non-degradative functions such as protein localization, DNA repair and regulation of chromatin structure. A small ubiquitin-related modifier (SUMO) uses a similar conjugation system that sometimes counteracts the effects of ubiquitination. Ubiquitin and SUMO compete for modification of proliferating cell nuclear antigen (PCNA), an essential processivity factor for DNA replication and repair. Whereas multi-ubiquitination is mediated by components of the RAD6 pathway and promotes error-free repair, SUMO modification is associated with replication. Here we show that RAD6-mediated mono-ubiquitination of PCNA activates translesion DNA synthesis by the damage-tolerant polymerases eta and zeta in yeast. Moreover, polymerase zeta is differentially affected by mono-ubiquitin and SUMO modification of PCNA. Whereas ubiquitination is required for damage-induced mutagenesis, both SUMO and mono-ubiquitin contribute to spontaneous mutagenesis in the absence of DNA damage. Our findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.     

RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO

The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

口腔鳞状细胞癌中错配修复基因hMLH1、hMSH2、p53和PCNA表达的相关性及意义

探讨hMLHI、hMSH2、p53和PCNA在OSCC中的表达关系及可能存在的临床意义。运用免疫组织化学S—P法对56例口腔鳞状细胞癌巾hMLH1、hMSH2、μ53和PCNA的表达进行检测。4种基因产物在OSCC中的阳性率均高于正常口腔黏膜,其中,中一低分化癌中的阳性率均高于高分化癌;hMSH2、p53和PCNA的阳性率在有淋巴结转移者中高于无转移者。hMLHI与p53／PCNA表达,hMSH2与p53／PCNA,p53与PCNA表达均呈正相关性。hM—LH1、hMSH2、p53和PCNA的异常表达及其相互之间的调节可能与OSCC的发生发展有关;检测4种蛋白有助于判断OSCC的恶性程度和生物学行为．     

Molecular structure and biological function of proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote. As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.     

Molecular structure and biological function of proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote. As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.     

PML contributes to p53-independent p21 up-regulation in gamma-irradiation induced DNA damage responses

The cyclin-dependent kinase inhibitor p21 WAF1/Cip1 is a critical cell cycle regulator which translocates into the nucleus to participate in DNA repair during DNA damage responses. In the present study, we showed that the tumor suppressor, promyelocytic leukemia protein (PML) contributes to the up-regulation of p21 in a p53-independent pathway. Knock-down of PML in p53-null H1299 and HCT 116 (p53 –/– ) tumor cells by specific siRNA resulted in down-regulation of p21 protein expression, inhibition of -irradi...     

Mouse p53 inhibits SV40 origin-dependent DNA replication

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.     

DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.     

SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase

Damaged DNA, if not repaired before replication, can lead to replication fork stalling and genomic instability; however, cells can switch to different damage bypass modes that permit replication across lesions. Two main bypasses are controlled by ubiquitin modification of proliferating cell nuclear antigen (PCNA), a homotrimeric DNA-encircling protein that functions as a polymerase processivity factor and regulator of replication-linked functions. Upon DNA damage, PCNA is modified at the conserved lysine residue 164 by either mono-ubiquitin or a lysine-63-linked multi-ubiquitin chain, which induce error-prone or error-free replication bypasses of the lesions. In S phase, even in the absence of exogenous DNA damage, yeast PCNA can be alternatively modified by the small ubiquitin-related modifier protein SUMO; however the consequences of this remain controversial. Here we show by genetic analysis that SUMO-modified PCNA functionally cooperates with Srs2, a helicase that blocks recombinational repair by disrupting Rad51 nucleoprotein filaments. Moreover, Srs2 displays a preference for interacting directly with the SUMO-modified form of PCNA, owing to a specific binding site in its carboxy-terminal tail. Our finding suggests a model in which SUMO-modified PCNA recruits Srs2 in S phase in order to prevent unwanted recombination events of replicating chromosomes.     

Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2

Ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers such as SUMO (also known as Smt3 in Saccharomyces cerevisiae) mediate signal transduction through post-translational modification of substrate proteins in pathways that control differentiation, apoptosis and the cell cycle, and responses to stress such as the DNA damage response. In yeast, the proliferating cell nuclear antigen PCNA (also known as Pol30) is modified by ubiquitin in response to DNA damage and by SUMO during S phase. Whereas Ub-PCNA can signal for recruitment of translesion DNA polymerases, SUMO-PCNA signals for recruitment of the anti-recombinogenic DNA helicase Srs2. It remains unclear how receptors such as Srs2 specifically recognize substrates after conjugation to Ub and Ubls. Here we show, through structural, biochemical and functional studies, that the Srs2 carboxy-terminal domain harbours tandem receptor motifs that interact independently with PCNA and SUMO and that both motifs are required to recognize SUMO-PCNA specifically. The mechanism presented is pertinent to understanding how other receptors specifically recognize Ub- and Ubl-modified substrates to facilitate signal transduction.     

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

5种细胞周期调控基因在小鼠生殖细胞发育过程中的表达研究

 以成熟(10周龄以上)的昆明种正常小鼠的精巢和卵巢为材料,利用地高辛标记的基因探针进行组织切片上的DNA-mRNA分子原位杂交,研究了PCNA,cdc2,cyclin D1,p2 1和 p16 5种细胞周期调控基因在生殖细胞发育过程中的表达.结果表明:PCNA基因在睾丸组织的精原细胞和精母细胞中有强杂交信号,而在雌性生殖细胞及滤泡细胞的发育过程都没有杂交信号;cyclin D1,cdc2,p2 1,p16基因在生殖细胞的发育过程中都没有,表明这些基因并没有参与小鼠生殖细胞的生长和分化调控.这些事实表明在生殖细胞发育过程中,控制细胞增殖和增殖抑制的基因与培养细胞有不同的机制,它们可能采用了不同的调控系统.     

A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage

The p53 gene is frequently inactivated in human cancers. Here we have isolated a p53-inducible gene, p53R2, by using differential display to examine messenger RNAs in a cancer-derived human cell line carrying a highly regulated wild-type p53 expression system. p53R2 contains a p53-binding sequence in intron 1 and encodes a 351-amino-acid peptide with striking similarity to the ribonucleotide reductase small subunit (R2), which is important in DNA synthesis during cell division. Expression of p53R2, but not R2, was induced by ultraviolet and gamma-irradiation and adriamycin treatment in a wild-type p53-dependent manner. Induction of p53R2 in p53-deficient cells caused G2/M arrest and prevented cells from death in response to adriamycin. Inhibition of endogenous p53R2 expression in cells that have an intact p53-dependent DNA damage checkpoint reduced ribonucleotide reductase activity, DNA repair and cell survival after exposure to various genotoxins. Our results indicate that p53R2 encodes a ribonucleotide reductase that is directly involved in the p53 checkpoint for repair of damaged DNA. The discovery of p53R2 clarifies a relationship between a ribonucleotide reductase activity involved in repair of damaged DNA and tumour suppression by p53.     

Role of poly(ADP-ribose) formation in DNA repair.

The abundant nuclear enzyme poly(ADP-ribose) polymerase catalyses the synthesis of poly(ADP-ribose) from nicotinamide adenine dinucleotide (NAD+). This protein has an N-terminal DNA-binding domain containing two zinc-fingers, which is linked to the C-terminal NAD(+)-binding domain by a short region containing several glutamic acid residues that are sites of auto-poly(ADP-ribosyl)ation. The intracellular production of poly(ADP-ribose) is induced by agents that generate strand interruptions in DNA. The branched homopolymer chains may attain a size of 200-300 residues but are rapidly degraded after synthesis. The function of poly(ADP-ribose) synthesis is not clear, although it seems to be required for DNA repair. Here we describe a human cell-free system that enables the role of poly(ADP-ribose) synthesis in DNA repair to be characterized. The results indicate that unmodified polymerase molecules bind tightly to DNA strand breaks; auto-poly(ADP-ribosyl)ation of the protein then effects its release and allows access to lesions for DNA repair enzymes.     

Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.