Abstract
The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR-Cas and other nucleases mediate double-strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template-independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non-homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.
Highlights
Introduction ingIn this mini-review, we provide a concise summary of the two known prokaryotic template-independent end joining pathways, Climate change, growing world population, and scarcity of re- and we elaborate on different strategies to employ clustered regusources are issues gaining increasing attention from society and larly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (CRISPR-Cas) systems and other nucleases in combination with these native or heterologously expressed DNAM
Being firstly predicted through in silico analyses, the prokaryotic non-homologous end joining (NHEJ) machinery was suggested to be considerably simpler than its eukaryotic counterpart, with only two proteins predicted to intervene, Ku and LigD.[50,51]
While NHEJ is more common in bacteria, it is considered rare in archaea, as not many species have the Ku protein;[48] so far, the full, canonical NHEJ system has only been described in the archaeal species Archaeoglobus fulgidus[50] and Methanocella paludicula.[52]
Summary
When a copy of the broken DNA is available, prokaryotes can repair double-strand DNA breaks with accuracy.[36]. Being firstly predicted through in silico analyses, the prokaryotic NHEJ machinery was suggested to be considerably simpler than its eukaryotic counterpart, with only two proteins predicted to intervene, Ku and LigD.[50,51] While NHEJ is more common in bacteria, it is considered rare in archaea, as not many species have the Ku protein;[48] so far, the full, canonical NHEJ system has only been described in the archaeal species Archaeoglobus fulgidus[50] and Methanocella paludicula.[52] Just like their eukaryotic homologs, prokaryotic Ku proteins form a ring-like structure that encloses broken DNA ends, protects them from the activity of cellular exonucleases and recruits LigD.[40] LigD, in turn, is a multidomain protein with nuclease, polymerase and (ATP-dependent) ligase activities, organization of which varies between species, and it can be present as a holoenzyme made of subunits.[47,53] Upon its recruitment by Ku, LigD processes the DNA ends with its nuclease and polymerase activities and ligates them in an ATP-dependent manner [54,55] (Figure 1A). A characteristic feature of AEJ is the large reliance on microhomologies (1–9 nucleotides), which are exposed due to the action of the RecBCD complex and enable DNA end annealing and ligation by the NAD-dependent DNA ligase A (LigA)[57] (Figure 1B)
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