DinB-RecA interaction is central to Escherichia coli's balance of genomic stability and adaptability through mutagenesis

• Online Access: http://hdl.handle.net/2047/D20316297

Maintaining genomic integrity is critical to cell survival, however it is constantly challenged by an onslaught of DNA damage by a variety of sources. To survive, bacteria induce a coordinated DNA damage response that helps the cell to strike a balance between preserving genomic integrity and adaptability through mutagenesis. The highly conserved Escherichia coli error-prone DNA polymerase IV, DinB, functions in several DNA damage tolerance and repair pathways. Its error-prone activity and high intracellular concentration give it a high potential for causing mutagenesis. It is critical for the cell to carefully control DinB's various functions to maintain the balance between genomic stability and adaptability. DinB performs translesion synthesis, the bypass of DNA lesions that would otherwise cause potentially lethal replication-fork stalling. Previous studies have determined that DinB's fidelity during translesion synthesis is enhanced upon interaction with RecA and that this activity is dependent on the highly conserved DinB residue cysteine 66. Here we find that this interaction results in a long-lasting conformational change in DinB. Interestingly this conformational change and DinB's enhanced fidelity persist beyond the time of interaction with RecA. This phenomenon may represent a novel regulatory mechanism to suppress DinB mutagenesis when it is not needed. DinB also plays a role in slowing DNA replication during the DNA damage response. This allows various DNA damage repair pathways to repair DNA lesions before they cause replication fork stalling. DinB can switch with the alpha subunit of DNA Polymerase III at an active replication fork and remodel the replisome to slow it down. Here we perform the first analysis of how DinB synthesizes using RNA primers, which exist on the lagging strand during DNA synthesis. We find that DinB synthesizes DNA inefficiently when using RNA primers and that interaction with RecA further inhibits this activity. This insight points to a novel mechanism for DinB to slow replication by accessing RNA primers on the lagging strand. DinB is the only translesion DNA polymerase that participates in the error-prone double strand break repair. During this process, DinB extends products of strand exchange, displacing one strand of dsDNA, while using the other strand as a template. This process introduces mutations into the genome due to DinB's error-prone synthesis. Structural modeling was used to identify a patch of amino acids that are highly conserved and located near the separating dsDNA during this type of synthesis. Notably, the residues in this patch are not conserved in other bacterial translesion DNA polymerases. We find that at least two of these conserved residues are critical for DinB's strand displacement activity, thus suggesting a structural explanation for why DinB is the only translesion polymerase that can perform this activity. In addition, we find that RecA assists DinB activity during strand displacement, which is an essential step in error-prone double strand break repair. These data point to a novel model that the DinB's interaction with RecA plays a role in maintaining the balance between genomic stability and adaptability through mutagenesis.