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.     

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.     

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.     

胆囊腺癌和腺瘤细胞核形态学计量和PCNA定量分析

研究胆囊腺瘤和腺瘤恶变与胆囊腺癌的关系。方法：用ＴＪＴＹ－３００全自动图像分析仪测定了２５例胆囊腺癌，２０例胆囊腺瘤和３例正常的胆囊上皮ＤＮＡ含量，并用ＡＢＣ免疫组化法对ＰＣＮＡ进行了测定。结果：（１）细胞核ＤＮＡ含量、异型性和ＰＣＮＡ指数在正常胆囊上皮－异型增生－腺瘤恶变－胆囊腺癌中逐渐增加；（２）胆囊腺瘤中异倍体比例与胆囊腺瘤的大小、合并胆囊结石有明显的关系（Ｐ＜００５），而与腺瘤数目、组织学类型无关。ＰＣＮＡ指数与腺瘤的大小有关，与腺瘤数目及组织学分类无关。结论：胆囊腺瘤具有明显的癌变潜能，腺瘤的直径≥１ｃｍ和伴有胆囊结石时癌变机率更大，应早期手术治疗     

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.     

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.     

大鼠肝再生中Cyclin D1、PCNA的动态变化

采用蛋白质印迹技术分析了肝切除不同恢复时期cyclin D1 和PCNA的含量变化,结果显示两者表达趋势基本一致,呈显著正相关(r=0.619**,P<0.01),提示cyclin D1 和PCNA在肝再生中细胞周期的 G1/S期转换和S 期DNA合成中发挥关键作用.     

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.     

A role for the nuclear envelope in controlling DNA replication within the cell cycle

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.     

组织芯片分析CD44v6和PCNA在口腔鳞癌及癌前病变中的表达及意义

目的：研究CDOv6和PCNA的表达与口腔鳞癌的发生、发展的关系。方法：应用组织芯片技术,通过免疫组织化学SP法分别检测一张组织芯片上117例组织样本中CD44v6、PCNA蛋白表达情况。结果：CD44v6在正常口腔黏膜组织和癌前病变呈阳性表达;癌组织中CD44v6的表达明显,但其表达程度随肿瘤恶性程度的增高而降低,PCNA的表达程度随着增生细胞和癌分级增加而升高。结论：在正常口腔黏膜上皮、增生上皮以及鳞癌中这两种蛋白的表达之间呈负相关。应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便、经济、准确的特点。     

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.     

CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth nor promotes DNA replication in the absence of CDK activity, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.     

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.     

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．     

人食管癌细胞增殖与超微弱发光

采用生物超微弱发光探测技术对食管癌细胞的超微弱发光进行研究,获得了食管癌细胞增殖与超微弱发光强度之间的关系.研究结果表明:不同密度组细胞的超微弱发光不同;超微弱发光与细胞的增殖状态有关.     

SD大鼠肝癌演进中PCNA及AZ的动态变化

目的:用二乙基亚硝胺(DENA)作诱变剂建立SD大鼠肝癌变各时期动物模型,探讨增殖核抗原(PC-NA)及抗酶(AZ)表达的动态变化.方法:从灌胃之日起,在2、4、6、8、10、12、14、16 w分8批处死大鼠,取其肝脏,进行病理分级、免疫组化和Western blot,对大鼠肝癌演进中细胞组织学变化及PCNA和AZ的...