DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts.
DNA polymerase epsilon (Pol epsilon) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases alpha and delta (Pol alpha and delta), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol epsilon in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol epsilon is indeed required for chromosomal DNA replication. In Pol epsilon-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol alpha are loaded onto the chromatin template in Pol epsilon-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol epsilon, along with Pol alpha and Pol delta, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.
Waga
S
S
Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. swaga@biken.osaka-u.ac.jp
Masuda
T
T
Takisawa
H
H
Sugino
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology; D016590:Aphidicolin / Q000494:pharmacology; D002457:Cell Extracts; D002467:Cell Nucleus / Q000235:genetics; D002843:Chromatin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002875:Chromosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D010063:Ovum / Q000201:enzymology*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D021122:Protein Subunits; D051716:Replication Protein A; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D013698:Templates, Genetic; D014982:Xenopus laevis / Q000235:genetics
D000906:Antibodies; D002457:Cell Extracts; D002843:Chromatin; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D051716:Replication Protein A; D016590:Aphidicolin; D004247:DNA; D004256:DNA Polymerase I; D004257:DNA Polymerase II