Insights into the molecular mechanism of replication fork rescue by RuvAB and the putative role of the bacterial RuvB-like protein RarA and its human homologue WRNIP1.
Doctoral thesis, UCL (University College London).
The RuvAB(C) proteins promote branch migration and resolution of Holliday junctions (HJs) which form during homologous recombination and upon reversal of stalled replication forks. In addition to the function of RuvAB to process HJs formed at stalled forks, RuvAB have recently been shown to be required to reverse stalled replication forks into HJs (chicken foot structures) in certain E. coli replication mutants in vivo (Baharoglu et al., 2008). We introduced two mutations in RuvA which disrupted interactions between tetramers and used the new mutant (RuvA2KaP) to investigate RuvAB function at Holliday junctions and stalled replication forks. Our data show that RuvA2KaP bound HJs and fork substrates as a single tetramer and at very high protein concentrations a second tetramer sandwiched the junction, but only if Mg2+ ions were present. The instability of RuvA2KaP octamer formation on HJ was demonstrated by the failure of RuvA2KaP to inhibit in vitro RuvC mediated junction resolution and in vivo RusA mediated resolution. RuvA2KaP promoted branch migration of HJ with RuvB but only at concentrations that also showed RuvA2KaP octamerization on HJ. The results directly showed that the stability of the RuvA2KaP RuvB complex on DNA was impaired in vitro. Studies in vivo showed that RuvA2KaP with RuvB could process Holliday junctions, based on its capacity to promote conjugational recombination and recombinational repair of UV induced damage. However, RuvA2KaP could not reverse replication forks. RuvA2KaP was therefore a clear separation-of-function mutant. In conclusion, the formation of a stable octamer of RuvA on stalled forks is required for fork reversal in vivo. RuvA2KaP was used to delineate the molecular cause of two other separation-of-function RuvA mutants: RuvAz3 and RuvAz87. In addition, RarA and its human homologue WRNIP1 were cloned, expressed and purified and these recombinant proteins were used to investigate the functions of RarA and WRNIP1 on DNA. WRNIP1 was also shown to localise in the nucleus in HeLa cells. Finally, the response of WRNIP1 to UV-induced damage was investigated; WRNIP1 foci accumulated in response to UV treatment of HeLa cells.
|Title:||Insights into the molecular mechanism of replication fork rescue by RuvAB and the putative role of the bacterial RuvB-like protein RarA and its human homologue WRNIP1|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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