Replication arrest and bypass at Tus/ter complexes in the terminus of the Escherichia coli chromosome
Kurakov, Anton Vladimirovich
Genome replication is frequently challenged by obstacles that can result from DNA damage, topological stress or tightly bound proteins. Replication fork stalling at DNA-bound proteins can lead to collapse of the fork and promote mutation and genomic instability, a hallmark of cancer cells. Interaction between replisomes and naturally occurring barriers can provide important information for understanding genome instability mechanisms. The simplicity of the Escherichia coli chromosome replication is ideal for studies of complex interactions between replication forks and replication barriers. E. coli carries a single chromosome that encodes a single origin of bidirectional replication, oriC, and a region diametrically opposite to oriC where replication terminates. The terminus region encodes four 23 bp ter sites, terA and terD on right replichore and terC and terB on the left. A ter sequence bound by Tus protein acts as a polar (unidirectional) natural barrier to fork progression. The Tus/ter system allows replisomes to enter the terminus, but will arrest their progress into the opposite replichore, that is, towards oriC. In this work two dimensional native-native gel electrophoresis was utilized to detect stalled replication forks at naturally occurring Tus/ter barriers in the E. coli chromosome terminus. The majority of arrested replication forks were found to accumulate at the first Tus/ter barrier on the left replichore, Tus/terC. Notably fewer arrested forks were detected at terA, terB and terD. The strength of ter sites was shown to be independent of the location in the terminus, whereas the sequence of ter sites was critical. The terB sequence forms the strongest terminator and restricts frequent replication fork bypass observed at terC. This correlates with the published data on the strength of nucleoprotein barriers formed by ter sequences observed in vitro. The presence of a strong terminator on each replichore helps the Tus/ter system prevent unwanted replication to escape the terminus. In the situation where additional rounds of replication were initiated in the terminus in the absence of the RecG helicase, most of replication forks were able to bypass Tus/terC barrier and were arrested at Tus/terB. Previous studies, in the Michel laboratory, revealed that the UvrD helicase can promote the bypass of a synthetically introduced Tus/terB replication fork barrier in the middle of the right replichore, but only as a consequence of RecA-mediated homologous recombination. The work presented in this thesis shows that, even in the absence of RecA-mediated homologous recombination, UvrD could promote the bypass of the naturally occurring Tus/terC (soft) barrier in the chromosome terminus. However, UvrD was unable to promote fork movement through stronger Tus/terB and Tus/terA barriers in the terminus. The terC and terB nucleotide sequences differ in three separate segments. I have shown that, one of the segments outside of the conserved region plays a critical role in the UvrD-dependent replication fork bypass of the Tus/terC barrier. My results suggest a distinct role of the UvrD helicase in the alleviation of replication fork stalling at the naturally occurring Tus/terC barrier in the chromosomal terminus.