Two Immunologically Distinct Human DNA Polymerase α-Primase Subpopulations Are Involved in Cellular DNA Replication

Dehde, Silke; Rohaly, Gabor; Schub, Oliver; Nasheuer, Heinz-Peter; Bohn, Wolfgang; Chemnitz, Jan; Deppert, Wolfgang; Dornreiter, Irena

doi:10.1128/mcb.21.7.2581-2593.2001

Cited by 33 publications

(45 citation statements)

References 43 publications

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“…This might represent an important regulatory mechanism for the recruitment of specific proteins to sites of DNA replication. For example, in mammalian cells two immunologically distinct pol-α-primase subpopulations have been identified (Dehde et al, 2001), which differ in the phosphorylation status of the p68 regulatory subunit of pol α. This post-translational modification is catalysed by the CDK2-cyclin-A complex.…”

Section: Pcna In Cell Cycle Controlmentioning

confidence: 99%

Proliferating cell nuclear antigen (PCNA): a dancer with many partners

Maga

Hübscher

2003

Journal of Cell Science

955

840

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Proliferating cell nuclear antigen (PCNA) was originally characterised as a DNA sliding clamp for replicative DNA polymerases and as an essential component of the eukaryotic chromosomal DNA replisome. Subsequent studies,however, have revealed its striking ability to interact with multiple partners, which are involved in several metabolic pathways, including Okazaki fragment processing, DNA repair, translesion DNA synthesis, DNA methylation,chromatin remodeling and cell cycle regulation. PCNA in mammalian cells thus appears to play a key role in controlling several reactions through the coordination and organisation of different partners. Two major questions have emerged: how do these proteins access PCNA in a coordinated manner, and how does PCNA temporally and spatially organise their functions? Structural and biochemical studies are starting to provide a first glimpse of how both tasks can be achieved.

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Section: Pcna In Cell Cycle Controlmentioning

confidence: 99%

Proliferating cell nuclear antigen (PCNA): a dancer with many partners

Maga

Hübscher

2003

Journal of Cell Science

955

840

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“…This antibody only recognizes the unmodified form of Mcm10. Anti-DNA polymerase-␣ (against the 180-kDa subunit) was a gift from Dr. Irene Dornreiter (Heinrich-PetteInstitut für Experimentelle Virologie and Immunologie, Universität Hamburg, Hamburg, Germany), and it was used at a 1:10 dilution for Western blotting (Dehde et al, 2001). Anti-p68 (against the B-subunit of DNA polymerase-␣) was a gift from Dr. Heinz-Peter Nasheuer (Heinz-Peter Nasheuer, Department of Biochemistry, National University of Ireland, Galway, Ireland), and it was used at a 1:3000 dilution for Western blotting.…”

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confidence: 99%

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

Chattopadhyay

Bielinsky

2007

MBoC

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In Saccharomyces cerevisiae, minichromosome maintenance protein (Mcm) 10 interacts with DNA polymerase (pol)-␣ and functions as a nuclear chaperone for the catalytic subunit, which is rapidly degraded in the absence of Mcm10. We report here that the interaction between Mcm10 and pol-␣ is conserved in human cells. We used a small interfering RNA-based approach to deplete Mcm10 in HeLa cells, and we observed that the catalytic subunit of pol-␣, p180, was degraded with similar kinetics as Mcm10, whereas the regulatory pol-␣ subunit, p68, remained unaffected. Simultaneous loss of Mcm10 and p180 inhibited S phase entry and led to an accumulation of already replicating cells in late S/G2 as a result of DNA damage, which triggered apoptosis in a subpopulation of cells. These phenotypes differed considerably from analogous studies in Drosophila embryo cells that did not exhibit a similar arrest. To further dissect the roles of Mcm10 and p180 in human cells, we depleted p180 alone and observed a significant delay in S phase entry and fork progression but little effect on cell viability. These results argue that cells can tolerate low levels of p180 as long as Mcm10 is present to "recycle" it. Thus, human Mcm10 regulates both replication initiation and elongation and maintains genome integrity. INTRODUCTIONDNA replication is a highly regulated process in which multiprotein complexes generate an exact copy of a cell's DNA. These multiprotein complexes are assembled into replication forks. Fork assembly takes place at replication origins that are spread throughout the genome in all eukaryotic cells. The first step is the formation of a prereplicative complex (pre-RC) (Diffley et al., 1994), which is subsequently transformed into a preinitiation complex, and finally into a pair of functional replication forks that have the ability to unwind parental DNA and synthesize new daughter strands (Mendez and Stillman, 2003). DNA unwinding is likely catalyzed by the minichromosome maintenance (Mcm) 2-7 complex (Aparicio et al., 1997;Labib et al., 2000;Shechter et al., 2004) in conjunction with two coactivators, Cdc45 and GINS (Pacek and Walter, 2004;Gambus et al., 2006;Moyer et al., 2006;Pacek et al., 2006). The association of Cdc45 and GINS with DNA is interdependent (Kubota et al., 2003;Takayama et al., 2003), and it requires yet another factor, Mcm10 (Wohlschlegel et al., 2002;Gregan et al., 2003;Sawyer et al., 2004). Despite its name, Mcm10 is not a member of the MCM protein family, although it was isolated in the same genetic screen in budding yeast that identified the MCM2-7 genes (Solomon et al., 1992;Merchant et al., 1997).Mcm10 is a constitutively nuclear DNA binding protein (Merchant et al., 1997;Burich and Lei, 2003) that is essential in yeast (Burich and Lei, 2003). Besides its role in DNA replication, Mcm10 has also been implicated in transcriptional silencing (Liachko and Tye, 2005). It is important to note that the general protein domain structure of Mcm10 is not unique in Saccharomyces cerevisiae, but it is highly con...

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“…Phosphorylation of p68/B subunit by cyclin-dependent kinases is observed in yeast and human cells at the G 1 /S transition and increases with progression to G 2 /M (11,18,20,49,52,60). However, B-subunit phosphorylation is delayed in yeast during the intra-S-phase checkpoint response in a Rad53-dependent manner (52).…”

Section: Discussionmentioning

confidence: 99%

“…The initiation of SV40 DNA replication in vitro is regulated by phosphorylation of pol-prim by cyclin/cdk (11,49,55,60,61). Phosphorylation of pol-prim by cyclin A/cdk2 on specific N-terminal residues of the p68 subunit, followed by purification of the pol-prim away from the kinase, resulted in a 10-fold inhibition of primer synthesis at the SV40 origin compared with mock-phosphorylated pol-prim (60,61).…”

Section: Resultsmentioning

confidence: 99%

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

Ott

Rehfuess

Podust

et al. 2002

Molecular and Cellular Biology

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DNA polymerase ␣-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.The recruitment of DNA polymerase ␣-primase (pol-prim) is a key event in the assembly of functional replication complexes in eukaryotic cells. pol-prim initiates DNA replication by synthesizing short RNA primers on the leading and lagging strand templates and then elongating them into hybrid primers of about 35 nucleotides (3,21,25,56,62,65). pol-prim is composed of four subunits that appear to be conserved in all eukaryotes. The human pol-prim subunits are named according to their apparent molecular weights: p180, p68, p58, and p48. The largest subunit contains the polymerase activity, and the smallest subunit contains the primase activity. Interactions of p68 with p180 and of p58 with p48 facilitate expression and nuclear import of the catalytic subunits (41,42). In addition, the p58 subunit physically links p48 to p180 and regulates the length of primers synthesized by the primase (3,4,7,10).The role of pol-prim in initiation of mammalian DNA replication has been investigated in the cell-free simian virus 40 (SV40) DNA replication system. In this model system, synthesis of RNA primers at the viral origin of replication requires only SV40 T antigen, the single-stranded DNA binding protein replication protein A (RPA), pol-prim, and topoisomerase I (23, 62). T antigen assembles on the viral origin, unwinds the parental DNA, and recruits the required cellular proteins to the replication fork. The single-stranded DNA (ssDNA) generated by T antigen is sequestered by RPA, and pol-prim synthesizes the first primers on the leading and lagging strand templates. Subsequently, pol-prim is displaced from the primer-template by the clamp-loader replication factor C (RFC), the PCNA sliding clamp, and DNA polymerase ␦, which extends the primers of both...

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Two Immunologically Distinct Human DNA Polymerase α-Primase Subpopulations Are Involved in Cellular DNA Replication

Cited by 33 publications

References 43 publications

Proliferating cell nuclear antigen (PCNA): a dancer with many partners

Proliferating cell nuclear antigen (PCNA): a dancer with many partners

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

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

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