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Wang, Mu,Park, Jang-Su,Ishiai, Masamichi,Hurwitz, Jerard,Lee, Suk-Hee 부산대학교 유전공학연구소 2000 분자생물학 연구보 Vol.16 No.-
Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication, we prepared heterologous RPAs containing the mixture of human and S. pombe subunits and compared these preparations for various enzmatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded(ss)DNA binding activity and an elongation of a primed DNa template catalyzed by DNA polymerase(pol) α and δ. A strong correlation between SV40 DNA replication activity and primer synthesis by pol α-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNa primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.
Cho, Won-Ho,Lee, Young-Joo,Kong, Soo-Im,Hurwitz, Jerard,Lee, Joon-Kyu National Academy of Sciences 2006 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.103 No.31
<P>Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ((5)S and (53)S) and at least three minor phosphorylation sites ((4)S, (7)S, and (59)T) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of (5)S and (53)S by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19-30 and containing (26)S and (27)S), it phosphorylated (26)S efficiently when this peptide contained a chemically synthesized phospho-(27)S modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclin-dependent and Cdc7 kinases is essential for the initiation of DNA replication.</P>
Im, Jun-Sub,Ki, Sang-Hee,Farina, Andrea,Jung, Dong-Soo,Hurwitz, Jerard,Lee, Joon-Kyu National Academy of Sciences 2009 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.106 No.37
<P>In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G(1)/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.</P>
Isolation of human Dna2 endonuclease and characterization of its enzymatic properties
Kim, Jeong-Hoon,Kim, Hee-Dai,Ryu, Gi-Hyuck,Kim, Do-Hyung,Hurwitz, Jerard,Seo, Yeon-Soo Oxford University Press 2006 Nucleic acids research Vol.34 No.6
<P>In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase δ-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of <I>Saccharomyces cerevisiae</I> Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5′ and 3′ tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5′ and 3′ regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells.</P>
The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing.
Kang, Young-Hoon,Kang, Min-Jung,Kim, Jeong-Hoon,Lee, Chul-Hwan,Cho, Il-Taeg,Hurwitz, Jerard,Seo, Yeon-Soo American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.16
<P>Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3' to 5' DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2Delta405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Delta405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5' to 3' exonuclease activity of Fen1. Mph1 binds to flap-structured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2Delta405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.</P>