Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase.
Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1(-/-) chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1(-/-) cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3(-/-) DT40 cells. Rather, we observed an increased number of replication fibers in Chk1(-/-) cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1(-/-) cells are associated with the accumulation of aberrant replication fork structures.
Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK. e.k.petermann@sussex.ac.uk
Eva
E
Petermann
Apolinar
A
Maya-Mendoza
George
G
Zachos
David A F
DA
Gillespie
Dean A
DA
Jackson
Keith W
KW
Caldecott
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002645:Chickens; D002875:Chromosomes; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D016196:S Phase; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D019311:Staurosporine