scholarly journals Human DNA polymerase delta is a pentameric holoenzyme with dimeric p12 subunit: Implications in enzyme architecture and PCNA interaction

2019 ◽  
Author(s):  
Prashant Khandagale ◽  
Doureradjou Peroumal ◽  
Kodavati Manohar ◽  
Narottam Acharya
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Essential Role ◽  
Amino Terminus ◽  
Dna Polymerase Delta ◽  
Human Dna ◽  
Evolutionarily Conserved ◽  
Carboxyl Terminal

AbstractHuman DNA polymerase delta (Polδ), a holoenzyme consisting of p125, p50, p68 and p12 subunits, plays an essential role in all the three DNA transaction processes. Herein, using multiple physicochemical and cellular approaches we found that the p12 protein forms a dimer in solution. In vitro reconstitution and pull-down of cellular Polδ by tagged p12 authenticates pentameric nature of this critical holoenzyme. Further, a consensus PIP motif at the extreme carboxyl terminal tail and a homodimerization domain at the amino-terminus of the p12 subunit were identified. Our mutational analyses of p12 subunit suggest that 3RKR5 motif is critical for dimerization that facilitates p12 binding to IDCL of PCNA via its PIP motif 98QCSLWHLY105. Additionally, we observed that oligomerization of the smallest subunit of Polδs is evolutionarily conserved as Cdm1 of S. pombe also dimerzes. Thus, we suggest that human Polδ is a pentameric complex with a dimeric p12 subunit; and discuss implications of p12 dimerization in regulating enzyme architecture and PCNA interaction during DNA replication.

scholarly journals Human DNA polymerase delta is a pentameric holoenzyme with a dimeric p12 subunit

2019 ◽  
Vol 2 (2) ◽  
pp. e201900323 ◽  
Author(s):  
Prashant Khandagale ◽  
Doureradjou Peroumal ◽  
Kodavati Manohar ◽  
Narottam Acharya
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Protein Motif ◽  
Human Dna ◽  
In Vitro Reconstitution ◽  
Evolutionarily Conserved ◽  
Carboxyl Terminal

Human DNA polymerase delta (Polδ), a holoenzyme consisting of p125, p50, p68, and p12 subunits, plays an essential role in DNA replication, repair, and recombination. Herein, using multiple physicochemical and cellular approaches, we found that the p12 protein forms a dimer in solution. In vitro reconstitution and pull down of cellular Polδ by tagged p12 substantiate the pentameric nature of this critical holoenzyme. Furthermore, a consensus proliferating nuclear antigen (PCNA) interaction protein motif at the extreme carboxyl-terminal tail and a homodimerization domain at the amino terminus of the p12 subunit were identified. Mutational analyses of these motifs in p12 suggest that dimerization facilitates p12 binding to the interdomain connecting loop of PCNA. In addition, we observed that oligomerization of the smallest subunit of Polδ is evolutionarily conserved as Cdm1 of Schizosaccharomyces pombe also dimerizes. Thus, we suggest that human Polδ is a pentameric complex with a dimeric p12 subunit, and discuss implications of p12 dimerization in enzyme architecture and PCNA interaction during DNA replication.

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex

1992 ◽  
Vol 12 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K Fien ◽  
B Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Replication Complex ◽  
Leading Strand ◽  
Factor C ◽  
Sv40 Dna

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

scholarly journals Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

1996 ◽  
Vol 16 (1) ◽  
pp. 94-104 ◽  
Author(s):  
F Stadlbauer ◽  
C Voitenleitner ◽  
A Brückner ◽  
E Fanning ◽  
H P Nasheuer
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Polymerase Alpha ◽  
Cell Extracts ◽  
Human Dna ◽  
Sv40 Dna ◽  
Hybrid Complexes

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.

scholarly journals Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

2017 ◽  
Vol 45 (16) ◽  
pp. 9427-9440 ◽  
Author(s):  
Dekang Liu ◽  
Jane H. Frederiksen ◽  
Sascha E. Liberti ◽  
Anne Lützen ◽  
Guido Keijzers ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Double Mutant ◽  
Dna Mismatch Repair ◽  
Dna Polymerase Delta ◽  
Dna Mismatch ◽  
Human Dna

scholarly journals Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Roland Chanet ◽  
Dorothée Baïlle ◽  
Marie-Pierre Golinelli-Cohen ◽  
Sylvie Riquier ◽  
Olivier Guittet ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Delta ◽  
Chromosomal Dna ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus

Abstract B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

scholarly journals Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

1992 ◽  
Vol 12 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K Fien ◽  
B Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Replication Complex ◽  
Leading Strand ◽  
Factor C ◽  
Sv40 Dna

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

scholarly journals Regulation and Modulation of Human DNA Polymerase δ Activity and Function

2017 ◽  
Author(s):  
Mariettta Y.W.T. Lee ◽  
Xiaoxiao Wang ◽  
Sufang Zhang ◽  
Zhongtao Zhang ◽  
Ernest Y.C. Lee.
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Delta ◽  
Cellular Functions ◽  
Human Dna ◽  
Delta Activity ◽  
Dna Replication And Repair ◽  
And Function ◽  
The Impact

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, PDIP46 and PDIP38, both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the splicesosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, Mdm2 alternative splicing and the regulation of the Nox4 NADPH oxidase.

scholarly journals A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

1995 ◽  
Vol 15 (8) ◽  
pp. 4420-4429 ◽  
Author(s):  
R Ayyagari ◽  
K J Impellizzeri ◽  
B L Yoder ◽  
S L Gary ◽  
P M Burgers
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Cold Sensitive ◽  
Factor C ◽  
Proliferating Cell

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

scholarly journals Role of the p68 Subunit of Human DNA Polymerase α-Primase in Simian Virus 40 DNA Replication

2002 ◽  
Vol 22 (16) ◽  
pp. 5669-5678 ◽  
Author(s):  
Robert D. Ott ◽  
Christoph Rehfuess ◽  
Vladimir N. Podust ◽  
Jill E. Clark ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Essential Role ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
Dna Polymerase Α

ABSTRACT DNA polymerase α-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

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