The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once
Introduction
The discovery of the double helix transformed biology and opened the doors for molecular biology and the field of genetics. However, DNA repair was not considered. Francis Crick wrote in 1974, “We totally missed the possible role of enzymes in repair although due to Claud Rupert's early very elegant work on photoreactivation, I later came to realize that DNA is so precious that probably many distinct repair mechanisms would exist.” [1]. DNA nucleases are essential players in DNA repair. For DNA, nucleases are a necessary evil. DNA damage needs to be trimmed off or removed, and this removal needs to be done both efficiently and accurately. Small errors in the substrate recognition or location of the incision can be deleterious to the cell and cause genomic instability. This review examines how nucleases ensure not only they have bound the correct substrate, but also that they do not bind and cut the wrong substrate. Here, we focus on DNA repair phosphoesterases that leave a 5′ phosphate and a 3′ hydroxyl suitable for polymerase extension and ligation. In particular, we analyze those whose structures have been determined with substrate and/or product DNA: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). There is now a sufficient number of enzymes meeting this criteria that useful insights emerge, and these insights have general importance. For the eukaryotic enzymes, we also include an examination of motifs that can be used to identify mechanistically similar nucleases. These enzymes are central to cell biology: they act in replication, base excision repair (BER), mismatch repair (MMR) double strand break repair (DSBR), and telomere maintenance. Furthermore they are increasingly found to act on RNA as well as DNA, and these activities may well be important as well.
Some of these nucleases are endonucleases that make a single cut within the DNA and some are exonucleases that processively cut from a DNA end, but some fall into both categories. The “restriction nuclease” discovered by Stuart Linn and Werner Arber [2], [3] provided breakthroughs in genetics because they provided enzymatic tools needed to “cut and paste” DNA molecules. Their specificity was based upon methylation or specific sequences, and thus they are site-specific nucleases. For damaged DNA, the discoveries of nucleotide excision repair and transcription-coupled repair pioneered by Phil Hanawalt and others sparked a dramatic evolution in our understanding of DNA and molecular biology by revealing the intriguing systems of DNA repair essential to life plus sets of nucleases needed for the cut-and-patch repair that are specific to DNA structure rather than sequence [4], [5], [6], [7], [8]. Thus, DNA damage repair nucleases have a different challenge than restriction nucleases with targeted sequences for incision. Although some recognize a modified base or phosphate backbone, others must recognize their substrates containing canonical nucleotides in an aberrant structure. The structure-specific nucleases in this review therefore provide a paradox of both extreme specificity and the lack of any sequence dependence with broad implications. For biotechnology, they can provide powerful tools to probe and modify DNA structure, as seen for FEN1 [9], [10]. Biochemically, if misregulated, they would destroy the integrity of genomic information. Biologically, they are necessary to preserving genome integrity and life itself.
How are these nucleases regulated? What is the basis for their exquisite specificity? Nuclease cutting is a committed step and thus tightly regulated. Structural biology provides key knowledge to address specificity questions and to contribute to a more complete and detailed understanding of their activities and biological functions. Particularly for these nucleases, structures furthermore provide detailed and rigorous information with which all other data should be reconciled and that often allows the integration of biochemical and genetic results. Examining the existing structures provides a basis to design mutants and inhibitors for separation of functions as seen for Mre11 [11], [12]. Yet, structures provide key knowledge not only to design mutations and inhibitors but also to interpret the impact of disease-causing mutations, as seen for XPD helicase [13], and the likelihood that polymorphisms may impact risks. As we come to understand DNA repair networks as more accurate than classical linear pathway concepts, we wish to control pathway choice and network crosstalk and interactions for biology and medicine. A detailed structural and mechanistic understanding of structure-specific nucleases, which is the focus of this review, is key to this goal. Increasingly we are finding that repair nuclease function requires changes in protein and DNA architecture that impacts binding, activity, and partner recruitment. Furthermore, flexible components (intrinsically unstructured regions) reshape or fold themselves in the presence of target DNA, as shown for FEN1 and its family members such as XPG [14], [15], [16], [17]. In essence these nucleases behave like molecular level transformers that can rebuild themselves by sometimes altering their protein conformations and typically sculpting the DNA to control both their specificity and efficiency functions. This knowledge suggests we need to re-think our understanding and the classic lock and key concept of how interactions, specificity, and activity are regulated with implications for inhibitor design.
Section snippets
Cell biology of DNA repair nucleases and increasing role as therapeutic targets
DNA repair nucleases permeate every DNA repair and processing pathway and are essential to the cell (Fig. 1). Damaged DNA can form spontaneously from endogenous metabolic sources, exogenously by DNA damaging agents (chemicals, radiation), or are intermediates from other repair or DNA processing enzymes. Damaged DNA must be incised from the DNA strand to prevent errors in coding or regulatory regions, to prevent mutations during replication, and to maintain genomic stability. Additionally,
Overview of DNA repair nuclease structures and mechanisms
Phosphodiesters are highly resistant to hydrolysis, with t1/2 of 30 million years at 25 °C [55]. Nucleases, such as FEN1, can accelerate that reaction 1017 fold [56]. Nucleases achieve this acceleration through a multistep acid–base reaction: (1) orientation of the attacking water for a linear attack on the phosphodiester bond; (2) activation of the attacking water through acid deprotonation; (3) stabilization of the electronegative pentacovalent intermediate, and (4) base protonation of the
Structurally-related family members: APE1, Nfo, TDP2 and UVDE
Abasic residues occur spontaneously or as intermediates in BER. Acting in the BER pathway, Endonuclease IV (EndoIV or Nfo) and apurinic/apyrimidinic endonuclease 1 (APE1) are two endonucleases that recognize abasic sites in the context of intact duplex DNA. Abasic sites can occur spontaneously or as repair intermediates from mono-functional or bi-functional glycosylases. Bi-functional glycosylases leave a 3′ deoxyribose, and APE1 and Nfo act as 3′ deoxyribonucleases to clean the 3′ end of these
Very short patch repair (Vsr) endonuclease
Microbial Vsr endonuclease recognizes and incises on the 5′ side of thymidine in TG mismatches in a very short patch repair process. Unlike the other enzymes discussed in this review, Vsr recognizes the damage within a specific sequence, CC*(A/T)GG that is the target for DNA-cytosine methyltransferase (Dcm). Spontaneous deamination of the second C (*) methylated by Dcm leads to the TG mismatch to be repaired. The overall structure of Vsr resembles type II restriction enzymes [86]. The
Endonuclease V (Nfi)
Nfi recognizes a surprisingly wide range of base damage in BER, including hypoxanthine, xanthine, oxanine, uracil, base mismatches, abasic sites, insertion/deletion loops, hairpins, and other aberrant DNA structures [92], [93], [94], [95], [96], [97], [98]. Nfi also incises RNA [99], [100]. This wide range of DNA damage recognized by Nfi means that it not only recognizes both damaged purines and pyrimidines, already distinct in size, but it also must recognize aberrant DNA structures with
5′ nuclease family: Flap endonuclease 1 (FEN1) and exonuclease 1 (Exo1)
FEN1 and Exo1 are part of the structure-specific 5′ nuclease superfamily that recognize ss-dsDNA junctions and cleave one nucleotide into the dsDNA. The members of this superfamily share two active site motifs in their sequences (Table 1). FEN1 incises 5′ flaps formed during Okazaki fragment maturation and during long patch BER. Exo1 is a processive 5′-3′ exonuclease that acts in MMR, DSBR, and telomere maintenance. The challenge of these 5′ nucleases in recognition of their structure-specific
RNaseT
A structure-specific nuclease that shares a similar steric wedge mechanism as the 5′ nucleases is RNase T. Although this DNA repair nuclease review has formally excluded RNases in its focus, Rnase T, despite its name, has been shown to have significant 3′-5′ exonuclease activity on ssDNA or 3′ overhangs with a 300-fold lower Km compared to RNA [108]. It has an unusual specificity, with its activity 100-fold reduced by a single C residue in the 3′ end [109]. Crystal structures with ss and 3′
Mre11
The dsDNA exonuclease and ssDNA endonuclease Mre11 recognizes, and processes double strand break (DSB) DNA ends. It also initiates the activation of the DNA-damage response through ATM. Furthermore, Mre11 degrades stalled replication forks [11], [110] where it also removes covalently bound topoisomerase [111]. Finally, Mre11 promotes micro-homology end joining at breaks during transcription [112]. With all these cellular functions, it is not surprising that Mre11 mutations are associated with
Integrating nuclease structures, chemistry, and biology
As a group, what do these structures inform us about the chemistry of the reaction and about the biology?
- (a)
Nucleases sculpt their substrate DNA to physically validate their substrates. Nucleases cannot see. Although this statement is obvious, we and other FEN1 researchers focused on the ssDNA, the most obvious visual difference between 5′ flaps and dsDNA. Instead, the structure revealed that FEN1 recognized that 5′ flap DNA had a break in the dsDNA and could bend >90° over a single
Synopsis and perspectives
This unified analysis of DNA repair nucleases reveals conserved themes in their mechanisms. The primary theme is the sculpting of the DNA with distortions, disruption of basepair stacking, flipped out nucleotides. These distortions are mediated near the active site through steric wedges that stick up in the path of the duplex DNA. Surprisingly, the structures of multiple nucleases with distinct mechanisms are teaching us that substrate specificity does not necessarily conform to the classic
Conflict of interest
The authors do not have a conflict of interest.
Acknowledgments
Work for this review and the authors′ efforts were funded by NIH RO1CA081967, RO1GM46312, and P01CA092584. J.L.V. is recipient of a fellowship from the Canadian Research Institutes of Health Research (CIHR).
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2021, Computational and Structural Biotechnology JournalCitation Excerpt :Thus, whereas sequence-based specificity explains part of the fidelity of DNA replication, key information is still missing about the mechanism controlling the precise accuracy by which the structure-based excision occurs. 5′ nucleases are highly conserved endo- and/or exo-nucleases that hydrolyze phosphodiester bonds situated 5′ end of ss/dsDNA junctions (Fig. 1A) [1–4]. This unified site of cleavage of diverse DNA structures is mediated by sharply bending the DNA at the ss/dsDNA junctions to position the scissile phosphate near the metal ions of the active site (Fig. 1B).