Disease relevance of POL30

• Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62) [1].
• Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli gamma complex in which only one subunit makes strong contact with the beta clamp [2].
• It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1 [3].
• The biochemical role of PCNA in rolling circle replication (RCR) of geminivirus DNA has not been explored in detail [4].

High impact information on POL30

• To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results [5].
• Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair [5].
• The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites [5].
• An msh6 mutation that eliminated the PCNA-binding site caused a mutator phenotype and a defect in the interaction with PCNA [6].
• Our results suggest that PCNA functions directly in mispair recognition and that mispair recognition requires a higher-order complex containing proteins in addition to Msh2p-Msh6p [6].

Biological context of POL30

• Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation [7].
• Unlike other REV genes, which encode DNA polymerases and an associated subunit, REV6 has been found to be identical to POL30, which encodes proliferating cell nuclear antigen (PCNA), the subunit of the homotrimeric sliding clamp, in which the rev6-1 mutation produces a G178S substitution [8].
• When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling [1].
• Our genetic studies with the pol30-119 mutation show that in addition to conferring a defect in Polzeta-dependent UV mutagenesis and in Poleta-dependent TLS, this PCNA mutation inhibits postreplicational repair of discontinuities that form in the newly synthesized strand across from UV lesions [9].
• Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA [10].

Anatomical context of POL30

• On a molar basis yPCNA and calf thymus PCNA/cyclin are equally active in stimulating DNA synthesis by DNA polymerase III [11].
• RAD18-independent ubiquitination of proliferating-cell nuclear antigen in the avian cell line DT40 [12].
• Proliferating cell nuclear antigen (PCNA) is the component of the chromosomal DNA replication machinery in eukaryotic cells that confers high processivity upon DNA polymerase delta and epsilon [13].
• As expected, DNA polymerase alpha and proliferating cell nuclear antigen showed relatively strong immunostaining in mitotically proliferating spermatogonia and even stronger staining in preleptotene cells undergoing meiotic DNA replication [14].
• In mouse and human fibroblasts, we found a significant positive correlation between the levels of SIRT1 and proliferating cell nuclear antigen (PCNA), a DNA processing factor expressed during S-phase [15].

Associations of POL30 with chemical compounds

• PCNA is also modified by SUMO conjugation at the lysine 164 residue [9].
• Here we report our studies with a proliferating cell nuclear antigen (PCNA) mutation, pol30-119, which results from a change of the lysine 164 residue to arginine [9].
• The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant [16].
• Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPgammaS) or ATP [17].
• Eliminating SUMO attachment to proliferating cell nuclear antigen or topoisomerase II does not affect doxorubicin cytotoxicity, whereas reducing SUMO attachment to the bud neck-associated septin proteins has a modest effect [18].

Physical interactions of POL30

• The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction [2].
• Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing [19].
• In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA [19].
• The POL32 gene product binds proliferating cell nuclear antigen [20].
• Two modes of FEN1 binding to PCNA regulated by DNA [21].

Enzymatic interactions of POL30

• Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair [22].

Regulatory relationships of POL30

• Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins [23].
• In addition, we provide evidence for the activation of the RAD52 recombinational pathway in the pol30-119 mutant and we infer that SUMO conjugation at the lysine 164 residue of PCNA has a role in suppressing the Rad52-dependent postreplicational repair pathway [9].
• In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA [24].
• Importantly, PCNA stimulates the 3'-->5' exonuclease and 3'-phosphodiesterase activities of Apn2 [25].
• Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA) [26].

Other interactions of POL30

• Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect [27].
• The PCNA-RFC families of DNA clamps and clamp loaders [1].
• The in vivo role of the PCNA interaction was investigated in the yeast Rad27 [16].
• We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta [28].
• The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumulate small single-stranded DNA fragments during DNA synthesis [29].

Analytical, diagnostic and therapeutic context of POL30

• The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis [30].
• Interactions of yeast RFC with PCNA and DNA were studied by surface plasmon resonance [17].
• However, when RFC and PCNA together were flowed across the DNA chip in the presence of ATP, a signal was observed suggesting loading of PCNA by RFC [17].
• Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae [13].

References