Jaylene N. Ollivierre

The Roles of UmuD in Regulating Mutagenesis

All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. Two E. coli Y-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms: UmuD2, which prevents mutagenesis, and UmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

Steric Gate Variants of UmuC Confer UV Hypersensitivity on Escherichia coli

Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the beta processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

The Dimeric SOS Mutagenesis Protein UmuD Is Active as a Monomer

The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage in Escherichia coli. UmuD2 is composed of 139-amino acid subunits and is up-regulated as part of the SOS response. Subsequently, damage-induced RecA·ssDNA nucleoprotein filaments mediate the slow self-cleavage of the N-terminal 24-amino acid arms yielding UmuD′2. UmuD2 and UmuD′2 make a number of distinct protein-protein contacts that both prevent and facilitate mutagenic translesion synthesis. Wild-type UmuD2 and UmuD′2 form exceptionally tight dimers in solution; however, we show that the single amino acid change N41D generates stable, active UmuD and UmuD′ monomers that functionally mimic the dimeric wild-type proteins. The UmuD N41D monomer is proficient for cleavage and interacts physically with DNA polymerase IV (DinB) and the β clamp. Furthermore, the N41D variants facilitate UV-induced mutagenesis and promote overall cell viability. Taken together, these observations show that a monomeric form of UmuD retains substantial function in vivo and in vitro.

Polymerase Switching in Response to DNA Damage

DNA polymerases are highly efficient and accurate macromolecular machines. They are capable of replicating DNA at up to 1,000 nucleotides per second while making less than one error in 100,000 additions. However, DNA is constantly subjected to damage from myriad sources. DNA damage disrupts normal cellular DNA replication by interfering with the accuracy and efficiency of replicative DNA polymerases. Specialized Y family DNA polymerases exist that can copy damaged DNA, although that ability often has a mutagenic cost. Therefore, Y family DNA polymerase activity is highly regulated in the cell. This chapter presents the functions of both replicative and Y family DNA polymerases and the cellular mechanisms of polymerase management. The focus is on Escherichia coli systems but also briefly discusses eukaryotic Y family polymerases. We first present DNA replication carried out by prokaryotic DNA polymerase III and describe its subunits and the coordination of leading and lagging strand replication. We then discuss DNA damage and specialized Y family DNA polymerases. Different models for the management of replicative and Y family DNA polymerases are presented. Finally, we briefly compare the eukaryotic systems with their prokaryotic counterparts.