Segregation of replicative DNA polymerases during S phase: DNA polymerase ε, but not DNA polymerases α/δ, are associated with lamins throughout S phase in human cells.
DNA polymerases (Pol) α, δ, and ε replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ε being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G(1)/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ε were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G(1)/S arrest and early in S phase, Pol α, δ, and ε were associated with the same nucleoprotein complexes, whereas in late S phase Pol ε and Pol α/δ were largely associated with distinct complexes. At G(1)/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ε, not Pol α/δ, remained associated with lamins. Consistently, Pol ε, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ε and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ε, to post-replicative processes such as translesion synthesis or post-replicative repair.
Department of Biology, University of Eastern Finland, Joensuu, Finland.
Markku
M
Vaara
Harri
H
Itkonen
Tomi
T
Hillukkala
Zhe
Z
Liu
Heinz-Peter
HP
Nasheuer
Daniel
D
Schaarschmidt
Helmut
H
Pospiech
Juhani E
JE
Syväoja
D002384:Catalysis; D002453:Cell Cycle; D047369:Chromatin Immunoprecipitation; D004256:DNA Polymerase I / Q000737:chemistry*; D004257:DNA Polymerase II / Q000737:chemistry*; D004258:DNA Polymerase III / Q000737:chemistry*; D004261:DNA Replication; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D034882:Lamins / Q000378:metabolism*; D009698:Nucleoproteins / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D016196:S Phase; D013347:Subcellular Fractions / Q000378:metabolism
D034882:Lamins; D009698:Nucleoproteins; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III