Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA.

Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the ß-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.

The Mycobacterium tuberculosis Ku C-terminus is a multi-purpose arm for binding DNA and LigD and stimulating ligation.

Bacterial non-homologous end joining requires the ligase, LigD and Ku. Ku finds the break site, recruits LigD, and then assists LigD to seal the phosphodiester backbone. Bacterial Ku contains a core domain conserved with eukaryotes but has a unique C-terminus that can be divided into a minimal C-terminal region that is conserved and an extended C-terminal region that varies in sequence and length between species. Here, we examine the role of Mycobacterium tuberculosis Ku C-terminal variants, where we removed either the extended or entire C-terminus to investigate the effects on Ku-DNA binding, rates of Ku-stimulated ligation, and binding affinity of a direct Ku-LigD interaction. We find that the extended C-terminus limits DNA binding and identify key amino acids that contribute to this effect through alanine-scanning mutagenesis. The minimal C-terminus is sufficient to stimulate ligation of double-stranded DNA, but the Ku core domain also contributes to stimulating ligation. We further show that wildtype Ku and the Ku core domain alone directly bind both ligase and polymerase domains of LigD. Our results suggest that Ku-stimulated ligation involves direct interactions between the Ku core domain and the LigD ligase domain, in addition to the extended Ku C-terminus and the LigD polymerase domain.

LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining.

DNA double-strand breaks are the most lethal form of damage for living organisms. The non-homologous end joining (NHEJ) pathway can repair these breaks without the use of a DNA template, making it a critical repair mechanism when DNA is not replicating, but also a threat to genome integrity. NHEJ requires proteins to anchor the DNA double-strand break, recruit additional repair proteins, and then depending on the damage at the DNA ends, fill in nucleotide gaps or add or remove phosphate groups before final ligation. In eukaryotes, NHEJ uses a multitude of proteins to carry out processing and ligation of the DNA double-strand break. Bacterial NHEJ, though, accomplishes repair primarily with only two proteins-Ku and LigD. While Ku binds the initial break and recruits LigD, it is LigD that is the primary DNA end processing machinery. Up to three enzymatic domains reside within LigD, dependent on the bacterial species. These domains are a polymerase domain, to fill in nucleotide gaps with a preference for ribonucleotide addition; a phosphoesterase domain, to generate a 3'-hydroxyl DNA end; and the ligase domain, to seal the phosphodiester backbone. To date, there are no experimental structures of wild-type LigD, but there are x-ray and nuclear magnetic resonance structures of the individual enzymatic domains from different bacteria and archaea, along with structural predictions of wild-type LigD via AlphaFold. In this review, we will examine the structures of the independent domains of LigD from different bacterial species and the contributions these structures have made to understanding the NHEJ repair mechanism. We will then examine how the experimental structures of the individual LigD enzymatic domains combine with structural predictions of LigD from different bacterial species and postulate how LigD coordinates multiple enzymatic activities to carry out DNA double-strand break repair in bacteria.