Regression supports two mechanisms of fork processing in phage T4.
Replication forks routinely encounter damaged DNA and tightly bound proteins, leading to fork stalling and inactivation. To complete DNA synthesis, it is necessary to remove fork-blocking lesions and reactivate stalled fork structures, which can occur by multiple mechanisms. To study the mechanisms of stalled fork reactivation, we used a model fork intermediate, the origin fork, which is formed during replication from the bacteriophage T4 origin, ori(34). The origin fork accumulates within the T4 chromosome in a site-specific manner without the need for replication inhibitors or DNA damage. We report here that the origin fork is processed in vivo to generate a regressed fork structure. Furthermore, origin fork regression supports two mechanisms of fork resolution that can potentially lead to fork reactivation. Fork regression generates both a site-specific double-stranded end (DSE) and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout infection. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway.
Long
David T
DT
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Kreuzer
Kenneth N
KN
D000677:Amsacrine / Q000494:pharmacology; D017122:Bacteriophage T4 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000821:virology; D006918:Hydroxyurea / Q000494:pharmacology; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D018741:Replication Origin / Q000187:drug effects; D014764:Viral Proteins / Q000378:metabolism
D014764:Viral Proteins; D000677:Amsacrine; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII; D006918:Hydroxyurea