Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes Most damage-induced mutagenesis in Escherichia coli is dependent upon the UmuD'C protein complex, which comprises DNA polymerase V (pol V). We recently discovered that pol V is also characterized by substantially reduced sugar selectivity. When the canonical Watson-Crick base pairing is preserved, purified pol V accompanied by accessory proteins, readily incorporates all ribonucleotides (ribonucleoside monophosphates, rNMPs) and catalyzes efficient and highly processive RNA synthesis in vitro in the presence of all four rNTPs. The ability of pol V to incorporate ribonucleotides is dramatically enhanced by a Y11A substitution at the conserved steric gate residue of UmuC. Interestingly, while UmuC_Y11A is highly inaccurate in vitro, it exhibits low mutability in vivo. Furthermore, despite the observation that the UmuC_Y11A variant catalyzed TLS past a T-T cyclobutane pyrimidine dimer (CPD) in vitro at least as efficiently as the wild-type enzyme, it conferred minimal UV-resistance to a delta umuDC strain. To explain these phenotypes, we suggested that the dramatic increase in rNMP incorporation promoted by UmuC_Y11A leads to the induction of downstream pathways involving rNMP processing. Specifically, that the rNMP-targeted repair pathways would not only reduce umuC_Y11A-dependent spontaneous and UV-induced mutagenesis, but also interfere with completion of TLS resulting in the observed decrease in UV resistance. The major enzymes initiating this pathway are ribonucleotide-specific endonucleases, Ribonuclease H (RNase H), which are present in organisms across all domains and are classified as types 1 and 2 based on sequence conservation and substrate preference. By taking advantage of the different capacities for ribonucleotide incorporation by the UmuC_Y11A pol V variant, we examined rNMP-processing pathways that cause phenotypic changes in strains expressing the pol V variants. While there was an 4-fold increase in the absolute number of Y11A-dependent mutations in the delta rnhA strain (lacking RNase HI) compared to the rnh+ strain, when expressed as a percentage of wild-type pol V-dependent mutagenesis, umuC_Y11A mutagenesis actually decreased from 7 to 5% of the wild-type levels. In contrast, in the isogenic delta rnhB strain (lacking RNase HII), the number of umuC_Y11A-dependent revertants increased approximately 5-fold compared to the rnhB+ strain and reached 40% of the level of mutagenesis observed with wild-type pol V. Our studies clearly demonstrated that RNase HII participates in a repair pathway that reduces the accumulation of rNMPs, as well as incorrect dNMPs incorporated into undamaged and damaged DNA by UmuC_Y11A. Based upon its in vitro properties, we expected pol V umuC_Y11A to be as mutagenic, if not more so, than the wild-type pol V, but even in the delta rnhB strain, Y11A-dependent mutagenesis was less than half of that observed with wild-type pol V, indicating that additional repair pathways act to reduce the mutagenic consequences of rNMPs incorporated by the highly error-prone umuC_Y11A. Indeed, in the isogenic delta rnhA delta rnhB strain umuC_Y11A spontaneous mutagenesis increased significantly to 72% of the level observed with wild-type pol V. Thus, our studies revealed that although RNase HI alone does not appear to participate in the removal of ribonucleotides incorporated by umuC_Y11A, in the absence of RNase HII, where there is likely to be a significant accumulation of ribonucleotides into DNA, RNase HI helps reduce the mutagenic burden of errant ribonucleotide incorporation into the E.coli genome. Our studies on the human TLS polymerases focused on DNA polymerase eta and iota. Both polymerases (pols) co-localize in replication factories in vivo after cells are exposed to UV-light and this co-localization is mediated through a physical interaction between the two TLS pols. The regions responsible for the interaction were previously loosely mapped to the C-terminal 200 amino acids of each protein. Although the two polymerases clearly co-localize at sites of DNA damage, the kinetics of their re-localization differs, suggesting that the two polymerases are not tightly associated in a living cell. Our studies begin to shed light on how such an interaction is facilitated and regulated. We identified the region in pol eta responsible for the interaction with pol iota as its ubiquitin binding zinc finger (UBZ) motif. Similarly, we demonstrated that the region responsible for the interaction between pol iota and pol eta is pol iotas ubiquitin binding motif (UBM). Since pol iota is also known to be monoubiquitinated in vivo, we hypothesized that the preferred partner of pol eta might actually be a ubiquitinated form of pol iota To test this hypothesis, we generated a chimera in which the N-terminus of ubiquitin was fused to the C-terminus of pol iota The mutant chimera lacked the two C-terminal glycine residues, and therefore only allowed for non-covalent interactions. The chimera interacted avidly with pol eta in the two-hybrid assays and this interaction was dependent upon I44 of ubiquitin (in the pol iota-Ub chimera). When expressed in human HEK293T cells the pol iota-Ub chimera was also able to pull-down considerably more pol eta than wild-type pol iota. We therefore concluded that the preferred partner for pol eta is a ubiquitinated form of pol iota The functional importance of the pol eta-iota interaction was clearly demonstrated by the fact that mutants of pol iota that are unable to interact with pol eta exhibit reduced accumulation of into replication factories. Conversely, the pol iota-Ub chimera, which exhibited a tighter interaction with pol eta showed an enhanced accumulation into replication foci. Given the complex set of protein-protein interactions that pol eta and pol iota are known to participate in, it seemed reasonable to predict that the ubiquitination status of the pols allows a cell a variety of ways to regulate the formation of TLS complexes. For example, monoubiquitination of pol eta is known to inhibit an interaction with ubiquitinated PCNA, but we demonstrated, it enhances its interaction with pol iota. Upon DNA damage, pol eta is de-ubiquitinated and this leads to a reduced ability to interact with pol iota, but a concomitant increased ability to interact with ubiquitinated PCNA, which helps explain why the polymerases exhibit different sub-cellular mobility in a living cell.

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