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

Abstract

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.

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

“…The entire C-terminus of M. tuberculosis Ku is also predicted by D 2 P 2 to be intrinsically disordered ( 35 ) (Figure 1C ), which is similar to models of B. subtilis Ku ( 12 , 13 ). However, in silico predictions of Ku by AlphaFold and ColabFold, suggest some structure within the M. tuberculosis Ku C-terminus ( 22 , 23 , 36 ). We cloned, expressed, and purified three Ku proteins based on the alignment in Figure 1B : (i) wildtype Ku (Ku WT ), (ii) Ku min , where the extended C-terminus is removed and only the minimal C-terminal region remains and (iii) Ku core , where the entire C-terminus is removed, leaving behind the conserved core domain.…”

“…From the sequence analysis, the minimal C-terminal region is conserved amongst bacteria, while the extended region varies in sequence and length, ranging from 1 to 155 amino acids, and is characteristically basic in nature ( 13 ). The structure of this C-terminus from SAXS modelling suggests a flexible, unstructured region ( 13 ), while in silico modelling suggests the C-terminus forms an alpha helix, with an additional disordered region ( 12 , 22 , 23 ).…”

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

“…Therefore, reconstitution of an efficient error-prone NHEJ pathway for repairing FnCas12a-generated DSBs in genome editing is more attractive since its application is convenient and can be scaled up when needed. The NHEJ machine in bacteria is well known that the Ku protein forms a homodimer, binds the initial break and recruits the multifunctional LigD to repair DSBs [37, 47]. Thus, we integrated the heterogenous NHEJ proteins (mtLigD and mtKu) from M. tuberculosis with our inducible CRISPR/FnCas12a nuclease system, and successfully achieved highly efficient knockout of the target effector genes in PXO99 A .…”

“…Three repair mechanisms have been reported for repairing the lethal DSBs in bacteria so far [27][28][29]. The HDR pathway is prevalent whereas the NHEJ pathway is available in only 20-25% of bacteria [47]. It leads to common use of homologous repair template in CRISPR-based bacterial genome editing, which achieves higher editing efficiency but still require the laborious and time-consuming cloning of homology arms and cannot apply in a large-scale.…”

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

“…Only two proteins, such as the DNA-end-binding protein Ku and a multifunctional ATP-dependent ligase (LigD), are required in the simplified NHEJ machine. In principle, homodimer Ku recognizes and binds to the broken DNA ends, recruiting LigD to rejoin the broken DNA ends in an error-prone way by direct ligation [31,32]. The alternative end joining mechanism (A-EJ; also referred to as microhomology-mediated repair, MMEJ) has been characterized in E. coli which lacks NHEJ, it largely relies on the action of RecBCD complex and Ligase-A in the presence of the microhomologies between DSB ends [33].…”