Bacterial NHEJ: a never ending story

Abstract

Double-strand breaks (DSBs) are the most detrimental DNA damage encountered by bacterial cells. DBSs can be repaired by homologous recombination thanks to the availability of an intact DNA template or by Non-Homologous End Joining (NHEJ) when no intact template is available. Bacterial NHEJ is performed by sets of proteins of growing complexity from Bacillus subtilis and Mycobacterium tuberculosis to Streptomyces and Sinorhizobium meliloti . Here, we discuss the contribution of these models to the understanding of the bacterial NHEJ repair mechanism as well as the involvement of NHEJ partners in other DNA repair pathways. The importance of NHEJ and of its complexity is discussed in the perspective of regulation through the biological cycle of the bacteria and in response to environmental stimuli. Finally, we consider the role of NHEJ in genome evolution, notably in horizontal gene transfer. Tel. +33 (0)3 72 74 51 43; Fax: +33 (0)3 83 68 44 99 # co-corresponding authors.

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Briefly, upon the formation of DSBs, dimeric complexes of Ku proteins bind to DSBs, and then recruit LigD to process the DNA termini, either resecting or synthesizing, or even reconstructing the original complementary ends. Finally, the break is sealed by LigD (Figure ) …”

“…Utilizing NHEJ process to repair the DSB delivered by Cas nuclease provides a striking feature that the homologous repair template is no longer required, which may significantly simplify the CRISPR‐based genome editing strategy in prokaryotes, enabling the construction of the single‐gene knockout library with substantially reduced labor. However, NHEJ mechanism is present in limited bacterial species, such as M. tuberculosis , M. smegmatis , B. subtilis , and Streptomyces species . A recent study in M. smegmatis demonstrated that the intrinsic NHEJ machines, Ku and LigD, worked efficiently to repair the DSB delivered by FnCpf1 in an error‐prone manner, giving an editing efficiency up to 70% for certain genes .…”

“…[139] However, NHEJ process was considered to be absent in prokaryotes for decades. [8,140,141] Until recently, bioinformatics analyses showed that only a few bacterial species hold Ku homologs. [142] These bacterial Ku proteins are usually clustered as an operon with an ATP-dependent DNA ligase (Ligase D, LigD), a multidomain protein functioning as polymerase, exonuclease, or ligase.…”

“…Finally, the break is sealed by LigD (Figure 4). [140,141] Utilizing NHEJ process to repair the DSB delivered by Cas nuclease provides a striking feature that the homologous repair template is no longer required, which may significantly simplify the CRISPR-based genome editing strategy in prokaryotes, enabling the construction of the single-gene knockout library with substantially reduced labor. However, NHEJ mechanism is present in limited bacterial species, such as M. tuberculosis, M. smegmatis, B. subtilis, and Streptomyces species.…”

“…However, NHEJ mechanism is present in limited bacterial species, such as M. tuberculosis, M. smegmatis, B. subtilis, and Streptomyces species. [69,141,143,145,146] A recent study in M. smegmatis demonstrated that the intrinsic NHEJ machines, Ku and LigD, worked efficiently to repair the DSB delivered by FnCpf1 in an error-prone manner, giving an editing efficiency up to 70% for certain genes. [69] However, the authors found that the intrinsic NHEJ in M. smegmatis did not cooperate with TdCas9_m or NmCas9 with unknown reason.…”