Crystal structure of RNase H3–substrate complex reveals parallel evolution of RNA/DNA hybrid recognition

Figiel, Małgorzata; Nowotny, Marcin

doi:10.1093/nar/gku615

Cited by 18 publications

(11 citation statements)

References 32 publications

(80 reference statements)

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“…RNase H enzymes incise the RNA strand of RNA:DNA hybrid duplexes; they are classified as type I (H1) or type II (H2 and H3) (14–17). RNase H1 requires an oligoribonucleotide tract and is unable to incise a single ribonucleotide embedded in duplex DNA.…”

Section: Introductionmentioning

confidence: 99%

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

et al. 2016

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RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

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Section: Introductionmentioning

confidence: 99%

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

et al. 2016

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“…The structures of RNA/DNA hybrids with different sequences have been studied alone (Benevides et al 1986;Fedoroff et al 1993) and in complex with different proteins (Rychlik et al 2010;Figiel and Nowotny 2014;Nishimasu et al 2014;Bernecky et al 2016). The results show that RNA/ DNA hybrids do not adopt the traditional B-conformation of DNA or A-conformation of RNA but occur as mixtures or heteromerous duplexes (Fedoroff et al 1993).…”

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confidence: 99%

Human proteins that interact with RNA/DNA hybrids

et al. 2018

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RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.

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“…RNase H enzymes incise the RNA strand of RNA:DNA hybrid duplexes; they are classified as type I (H1) or type II (H2 and H3) (16)(17)(18)(19). RNase H1 requires an oligoribonucleotide tract and is unable to incise a single ribonucleotide embedded in duplex DNA.…”

mentioning

confidence: 99%

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

Jacewicz

Shuman

2015

J Bacteriol

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Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3=-OH end and at least one or two ribonucleotides on the 5=-PO 4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. IMPORTANCERNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. RnhC has a C-terminal acid phosphatase domain that is functionally autonomous of its N-terminal RNase H catalytic domain. RnhC homologs are prevalent in Actinobacteria.T he human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis have a large roster of DNA repair enzymes, including a shared set of eight DNA polymerases (1-5), four of which-LigD-POL, PolD1, PolD2, and DinB2-have the distinctive properties of low fidelity and readily incorporating ribonucleotides in lieu of deoxyribonucleotides during primer extension and gap repair in vitro (4-10). LigD-POL, PolD1, and PolD2 are paralogous members of the AEP (archaeal/eukaryal polymerase/primase) polymerase family (4, 10, 11). They incorporate between one and four sequential ribonucleoside monophosphates (rNMPs) at a DNA primer terminus; after four rNMPs, they cease to elongate (4,8). This effect is attributed to their inability to extend an RNA:DNA hybrid primer terminus with a fully A-form helical conformation (8). DinB2 is a Y family polymerase, and it...

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Crystal structure of RNase H3–substrate complex reveals parallel evolution of RNA/DNA hybrid recognition

Cited by 18 publications

References 32 publications

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

Human proteins that interact with RNA/DNA hybrids

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

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