London Research Institute
-Clare Hall Laboratories-
-Cancer Research UK-
Charity Registration No. 1089464 -Terms and Conditions-
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Molecular Enzymology
London Research Institute -Clare Hall Laboratories- -Cancer Research UK- Charity Registration No. 1089464 -Terms and Conditions- |
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| Structural Studies on the RecG Protein |
Chromosomal replication in prokaryotes has long been described in terms of initiation from a single origin, followed by bidirectional replication of the chromosome until a termination sequence is reached. However, it has become apparent1 that the replication fork rarel progresses unimpeded from origin to terminator. Indeed, it is likely that all replication forks stall at some point during the replicative process as a result of encountering various forms of damage in the DNA template. These problems necessitate a pathway or pathways for repairing or avoiding the damaged DNA and restarting the stalled fork. One such mechanism for damage bypass involves the protein RecG. Biochemical2 and genetic3 studies have shown that the protein is able to convert stalled forks into so-called 'chicken-foot' structures in an ATP-dependent manner (Figure 1). 
This chicken-foot structure is a suitable substrate for a process known as template switching, first proposed in 19764. This allows a polymerase to bypass a lesion in one strand of the DNA, by using the newly-synthesized complementary daughter strand as a template. The four-way (Holliday) junction formed by this process may then be migrated, for example by the RuvAB proteins, to re-establish the replication fork with the DNA lesion bypassed, for later repair. This process is shown schematically in the following movie.
Animation of template switching and lesion bypass (automatically restarts).
The sequence of events shown are 1) fork progression until a lesion is encountered, 2) asymmetric continuation of synthesis on the lagging (blue) strand, 3) unwinding of the nascent lagging strand and reversal of fork movement, 4) synthesis of new strand (green) from end of prematurely terminated leading strand (red), 5) the newly formed four-way junction is migrated until the end of the newly synthesised strand leading to 6) the re-establishment of the fork. We decided to try and determine the structure of RecG in complex with a DNA substrate to elucidate the molecular mechanism of this process.
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