Structure of a Preternary Complex Involving a Prokaryotic NHEJ DNA Polymerase

Brissett, Nigel C.; Martín, María J.; Pitcher, R. S.; Bianchi, Julie; Juárez, Raquel; Green, Andrew J.; Fox, Gavin C.; Blanco, Luis; Doherty, Aidan J.

doi:10.1016/j.molcel.2010.12.026

Cited by 42 publications

(81 citation statements)

References 27 publications

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“…Pol may ingress into the termini to uncover regions of microhomology that then can be annealed together to facilitate end synapsis (24). It also may enable these enzymes to locate an internal 5′ phosphate, if a terminal one is not present, to allow it to bind more securely to a DSB (24,28). However, although it may be beneficial for some purposes, strand displacement can lead to some undesirable consequences, as discussed below.…”

Section: Mpa Nhej Proteins Possess Activities Equivalent To Those Of mentioning

confidence: 99%

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Bartlett

Brissett

Doherty

2013

Proc. Natl. Acad. Sci. U.S.A.

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Nonhomologous end-joining (NHEJ) pathways repair DNA doublestrand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3′ ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3′ phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3′ monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE's function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3′ ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair. D NA double-strand breaks (DSBs) are one of the most lethal forms of damage encountered by cells (1). Nonhomologous end joining (NHEJ) is the primary pathway for repairing DSBs in higher eukaryotes (2). NHEJ does not require the presence of a sister chromatid, in contrast to homologous recombination, and therefore can operate in quiescent cells. The direct repair and ligation of DSBs by NHEJ is considered to be more error prone because breaks are mended without the assistance of an intact DNA template.The higher eukaryotic NHEJ complex is composed primarily of the ligase IV, X-ray repair cross-complementing protein 4 (XRCC4), and XRCC4-like factor (XLF) complex (the LXX complex), DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), and Ku70/80 (1, 3). Ku and DNA-PKcs both assist in bridging the gap between the broken termini by promoting end synapsis. NHEJ repair of nonhomologous breaks requires remodeling of the DNA termini by processing enzymes, including polymerases (Pol λ and μ) and nucleases [Artemis and flap endonuclease 1 (Fen-1)], to prepare them for ligation (4-7). The LXX complex is recruited by Ku, which enables the ligation of the DNA ends.Most bacterial organisms also possess an NHEJ complex, composed of a more limited set of proteins, including ligase D (LigD) and Ku (8,9), which is required for DSB repair in stationary phase (10). The mycobacterial LigD gene encodes a multifunctional enzyme which, along with Ku, is ...

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Section: Mpa Nhej Proteins Possess Activities Equivalent To Those Of mentioning

confidence: 99%

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Bartlett

Brissett

Doherty

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…3). In polymerases, such as the polymerase domain (PolDom) of the NHEJ DNA polymerase of M. tuberculosis, an arginine residue is also present (Brissett et al, 2011). This arginine interacts with one aspartate residue of the above-mentioned acidic triad, resulting in what is called the ''pre-ternary'' complex.…”

Section: The Head Domain Of Csn2 Exhibits a Helicase Foldmentioning

confidence: 99%

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

Ellinger

Arslan

Wurm

et al. 2012

Journal of Structural Biology

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“…LigD proteins often consist of an ATP-dependent DNA ligase domain (LIG), a polymerase domain (PolDom or POL), and a 3 -phosphoesterase domain (PE), called also as a nuclease domain, that could account for the DNA ends processing, gap filling and sealing steps in NHEJ [15,16,18,23,[25][26][27][28][29][30][31][32][33]. POL domain catalyses either non-templated single nucleotide additions to a blunt-ended duplex DNA (primase activity) or possesses DNA-dependent DNA/RNA gap filling polymerase activities [18,[23][24][25]27].…”

Section: Introductionmentioning

confidence: 99%

NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida

et al. 2015

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Structure of a Preternary Complex Involving a Prokaryotic NHEJ DNA Polymerase

Cited by 42 publications

References 27 publications

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida

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