naomi huang - Academia.edu (original) (raw)

Papers by naomi huang

The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nu... more The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides (rNMPs) from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMPassociated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease. The exclusion and removal of rNMPs from DNA are important for the stability and function of the genome. In Saccharomyces cerevisiae, the introduction of an rNMP-permissive form of DNA polymerase ε into a strain lacking RNase H2 confers a mutator phenotype and is associated with the accumulation of a distinct mutation class: deletions within short (2-5 bp) tandem repeats (1). In contrast to similar mutations initiated by DNA polymerase slippage during genome replication, however, the rNMP-associated deletion intermediates are not substrates for the postreplicative mismatch repair machinery (2). A similar deletion signature is associated with high levels of transcription in yeast and requires the activity of Top1 (3, 4), a type 1B topoisomerase important for removing transcription-associated supercoils (5). Here, we demonstrate that rNMP-associated deletions are likewise dependent on Top1 activity, map in vitro the positions of Top1 cleavage at deletion hotspots identified in vivo, and confirm that Top1 has endoribonuclease activity when an rNMP is substituted at the scissile phosphate. The CAN1 gene encodes arginine permease, the loss of which confers resistance to the toxic arginine analog canavanine (Can-R phenotype). To determine the effect of persistent rNMPs on CAN1 mutagenesis in yeast, we deleted the RNH201 gene, which encodes the catalytic subunit of RNase H2 (6). While there was only a small elevation in the Can-R rate in the rnh201 background, there was a substantial change in the corresponding mutation spectrum, with ~40% of mutations being deletions of 2-5 bp (partial and complete spectra are presented in Fig. 1 and Fig. S1, respectively). To examine whether Top1 activity is relevant to rNMP-associated mutagenesis, we deleted the TOP1 gene from the rnh201 background. The rate of short deletions in the double mutant reverted to that observed in the WT strain, demonstrating that Top1 is required for the rNMP-associated deletion signature. Hereafter, we focus on the 2-bp deletion class, which localizes to discrete hotspots that coincide with

The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nu... more The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides (rNMPs) from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMPassociated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease. The exclusion and removal of rNMPs from DNA are important for the stability and function of the genome. In Saccharomyces cerevisiae, the introduction of an rNMP-permissive form of DNA polymerase ε into a strain lacking RNase H2 confers a mutator phenotype and is associated with the accumulation of a distinct mutation class: deletions within short (2-5 bp) tandem repeats (1). In contrast to similar mutations initiated by DNA polymerase slippage during genome replication, however, the rNMP-associated deletion intermediates are not substrates for the postreplicative mismatch repair machinery (2). A similar deletion signature is associated with high levels of transcription in yeast and requires the activity of Top1 (3, 4), a type 1B topoisomerase important for removing transcription-associated supercoils (5). Here, we demonstrate that rNMP-associated deletions are likewise dependent on Top1 activity, map in vitro the positions of Top1 cleavage at deletion hotspots identified in vivo, and confirm that Top1 has endoribonuclease activity when an rNMP is substituted at the scissile phosphate. The CAN1 gene encodes arginine permease, the loss of which confers resistance to the toxic arginine analog canavanine (Can-R phenotype). To determine the effect of persistent rNMPs on CAN1 mutagenesis in yeast, we deleted the RNH201 gene, which encodes the catalytic subunit of RNase H2 (6). While there was only a small elevation in the Can-R rate in the rnh201 background, there was a substantial change in the corresponding mutation spectrum, with ~40% of mutations being deletions of 2-5 bp (partial and complete spectra are presented in Fig. 1 and Fig. S1, respectively). To examine whether Top1 activity is relevant to rNMP-associated mutagenesis, we deleted the TOP1 gene from the rnh201 background. The rate of short deletions in the double mutant reverted to that observed in the WT strain, demonstrating that Top1 is required for the rNMP-associated deletion signature. Hereafter, we focus on the 2-bp deletion class, which localizes to discrete hotspots that coincide with

The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nu... more The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides (rNMPs) from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMPassociated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease. The exclusion and removal of rNMPs from DNA are important for the stability and function of the genome. In Saccharomyces cerevisiae, the introduction of an rNMP-permissive form of DNA polymerase ε into a strain lacking RNase H2 confers a mutator phenotype and is associated with the accumulation of a distinct mutation class: deletions within short (2-5 bp) tandem repeats (1). In contrast to similar mutations initiated by DNA polymerase slippage during genome replication, however, the rNMP-associated deletion intermediates are not substrates for the postreplicative mismatch repair machinery (2). A similar deletion signature is associated with high levels of transcription in yeast and requires the activity of Top1 (3, 4), a type 1B topoisomerase important for removing transcription-associated supercoils (5). Here, we demonstrate that rNMP-associated deletions are likewise dependent on Top1 activity, map in vitro the positions of Top1 cleavage at deletion hotspots identified in vivo, and confirm that Top1 has endoribonuclease activity when an rNMP is substituted at the scissile phosphate. The CAN1 gene encodes arginine permease, the loss of which confers resistance to the toxic arginine analog canavanine (Can-R phenotype). To determine the effect of persistent rNMPs on CAN1 mutagenesis in yeast, we deleted the RNH201 gene, which encodes the catalytic subunit of RNase H2 (6). While there was only a small elevation in the Can-R rate in the rnh201 background, there was a substantial change in the corresponding mutation spectrum, with ~40% of mutations being deletions of 2-5 bp (partial and complete spectra are presented in Fig. 1 and Fig. S1, respectively). To examine whether Top1 activity is relevant to rNMP-associated mutagenesis, we deleted the TOP1 gene from the rnh201 background. The rate of short deletions in the double mutant reverted to that observed in the WT strain, demonstrating that Top1 is required for the rNMP-associated deletion signature. Hereafter, we focus on the 2-bp deletion class, which localizes to discrete hotspots that coincide with

The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nu... more The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides (rNMPs) from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMPassociated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease. The exclusion and removal of rNMPs from DNA are important for the stability and function of the genome. In Saccharomyces cerevisiae, the introduction of an rNMP-permissive form of DNA polymerase ε into a strain lacking RNase H2 confers a mutator phenotype and is associated with the accumulation of a distinct mutation class: deletions within short (2-5 bp) tandem repeats (1). In contrast to similar mutations initiated by DNA polymerase slippage during genome replication, however, the rNMP-associated deletion intermediates are not substrates for the postreplicative mismatch repair machinery (2). A similar deletion signature is associated with high levels of transcription in yeast and requires the activity of Top1 (3, 4), a type 1B topoisomerase important for removing transcription-associated supercoils (5). Here, we demonstrate that rNMP-associated deletions are likewise dependent on Top1 activity, map in vitro the positions of Top1 cleavage at deletion hotspots identified in vivo, and confirm that Top1 has endoribonuclease activity when an rNMP is substituted at the scissile phosphate. The CAN1 gene encodes arginine permease, the loss of which confers resistance to the toxic arginine analog canavanine (Can-R phenotype). To determine the effect of persistent rNMPs on CAN1 mutagenesis in yeast, we deleted the RNH201 gene, which encodes the catalytic subunit of RNase H2 (6). While there was only a small elevation in the Can-R rate in the rnh201 background, there was a substantial change in the corresponding mutation spectrum, with ~40% of mutations being deletions of 2-5 bp (partial and complete spectra are presented in Fig. 1 and Fig. S1, respectively). To examine whether Top1 activity is relevant to rNMP-associated mutagenesis, we deleted the TOP1 gene from the rnh201 background. The rate of short deletions in the double mutant reverted to that observed in the WT strain, demonstrating that Top1 is required for the rNMP-associated deletion signature. Hereafter, we focus on the 2-bp deletion class, which localizes to discrete hotspots that coincide with