Report 2008-2010

Figure 4. Comparison of a MTB ImuA’ (Rv3395c) model with the RecA crystal structure. ImuA’ appears to be a “crippled” RecA homolog. It
lacks the C-terminal subdomain and ATP-binding site.
Previously, these genes were identified as putative mycobacterial components
of an imuA’-imuB-dnaE2 mutagenic cassette widespread in
bacteria. Using computational techniques we have generated models
for all three Mtb proteins. Combining these models with sequence
analysis we showed that although Rv3394c is related to Y-family DNA
polymerase, it cannot function as a polymerase, because it lacks the
essential active site residues. The computational analysis also identified
a substantial disorder within the C-terminal region, suggesting its
participation in protein-protein interactions. The subsequent experiments
in the Prof. V. Mizrahi’s lab directly confirmed computational
findings. In turn, a computational model of Rv3395c (Fig. 5) allowed
the design of its truncation variants that subsequently were used to
map the Rv3395c regions important for its interaction with Rv3394c.
A computational model of the DnaE2 polymerase, which was shown
to be directly responsible for the mutagenic translesion synthesis (TLS),
provided clues as to its functional specialization. Compared with the
main replicative polymerase (DnaE1), DnaE2 lacks the C-terminal domain,
which mediates interaction with the polIII t-subunit. The t-subunit
is coordinating leading and lagging DNA strand synthesis during
replication. The absence of the t-interacting domain suggests that
DnaE2 is unable to substitute DnaE1 within the replisome. In addition,
the absence of key histidine and aspartate residues in the PHP domain
of DnaE2 hints at compromised proofreading function. Taken together,
these computational results played an important role in revealing individual
roles of the DnaE2-Rv3394c-Rv3395c “mutagenic complex”
components as well as mapping their interaction regions. Results of
the study have been reported in Proceedings of the National Academy
of Sciences of the USA [6].
Rv3394c
S.Solfataricus Dpo4
Figure 5. Structurally, MTB ImuB (Rv3394c) is very similar to a Y-family polymerase (Dpo4), but it lacks polymerase active site residues.
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