Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII<sup>1</sup>

Frank, Peter; Braunshofer-Reiter, Christa; Wintersberger, Ulrike

doi:10.1016/s0014-5793(97)01528-7

Cited by 52 publications

(46 citation statements)

References 30 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In vivo, FEN1-directed RNA removal may be only one of several redundant pathways. Previous studies indicated that RNase H is responsible for RNA primer removal (42), but recent genetic studies show that the deletion of the RNase H catalytic subunit did not affect cell viability in yeast (43). This suggests that other enzymes are also involved in RNA removal, one of which is Dna2p.…”

Section: Discussionmentioning

confidence: 98%

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Kao

Henricksen²,

Liu

et al. 2002

Journal of Biological Chemistry

139

161

View full text Add to dashboard Cite

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3-tail on the upstream primer adjacent to the 5-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.Pathways for DNA replication, recombination, and repair are all proposed to involve DNA flap intermediates (1-8). The creation and resolution of single-stranded flap structures is a preferred method for removal of damaged or mismatched nucleotides. DNA replication requires the synthesis and joining of discontinuous segments, or Okazaki fragments. Each fragment is initiated by an RNA primer that must be removed prior to joining. Synthesis from the upstream fragment is thought to displace the RNA primer and some adjacent DNA into a single-stranded flap (8 -11). Processing of the flap intermediate is carried out by the structure-specific flap endonuclease 1 or FEN1 1 (4).FEN1 is evolutionarily conserved, and it has intrinsic 5Ј-3Ј-exonuclease and endonuclease activities (4,5,(12)(13)(14). The exonuclease activity utilizes double-stranded DNA with a nick or gap, whereas the endonuclease activity requires a flap structure. In prokarya, the FEN1 homologue is the 5Ј-nuclease domain associated with DNA polymerase I; however, FEN1 exists as a separate polypeptide in eukarya, archaea, and some bacteriophages (15). The mechanism and substrate specificity of FEN1 have been studied by many groups (4, 15-23). The preferred substrate for endonucleolytic cleavage was defined as a nick-flap substrate in vitro. It consists of upstream and downstream primers annealed to a template in a manner that creates a nick. The downstream primer contains a single-stranded 5Ј-flap region (4, 24).Sever...

show abstract

Section: Discussionmentioning

confidence: 98%

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Kao

Henricksen²,

Liu

et al. 2002

Journal of Biological Chemistry

139

161

View full text Add to dashboard Cite

show abstract

“…In contrast, more recent studies have indicated that the major in vitro mechanism for RNA primer removal is flap processing by Rad27/FEN1 and Dna2 (14)(15)(16), with Exo1 apparently being able to substitute for Rad27/FEN1 in S. cerevisiae (17,18). Because RNase H2 is not essential in S. cerevisiae (19), it is possible that RNase H2 plays either a redundant or a stimulatory role in Okazaki fragment processing, which could underlie the substantial growth defects caused by combining mutations affecting RNase H2 with mutations affecting Rad27/Fen1 (7,(20)(21)(22)(23).…”

mentioning

confidence: 98%

A Saccharomyces cerevisiae RNase H2 Interaction Network Functions To Suppress Genome Instability

et al. 2014

View full text Add to dashboard Cite

Errors during DNA replication are one likely cause of gross chromosomal rearrangements (GCRs). Here, we analyze the role of RNase H2, which functions to process Okazaki fragments, degrade transcription intermediates, and repair misincorporated ribonucleotides, in preventing genome instability. The results demonstrate that rnh203 mutations result in a weak mutator phenotype and cause growth defects and synergistic increases in GCR rates when combined with mutations affecting other DNA metabolism pathways, including homologous recombination (HR), sister chromatid HR, resolution of branched HR intermediates, postreplication repair, sumoylation in response to DNA damage, and chromatin assembly. In some cases, a mutation in RAD51 or TOP1 suppressed the increased GCR rates and/or the growth defects of rnh203⌬ double mutants. This analysis suggests that cells with RNase H2 defects have increased levels of DNA damage and depend on other pathways of DNA metabolism to overcome the deleterious effects of this DNA damage.

show abstract

“…In addition, RNase HI may be involved in Okazaki fragment processing, since in vitro it preferentially cleaves 5Ј to the last ribonucleotide of an RNA-DNA junction to remove an RNA primer (9,62). Rnh35, the catalytic subunit of the yeast homolog of mammalian RNase HI, may work cooperatively with Rad27 to remove RNA primers, since Rad27 preferentially cleaves a single-stranded flap of RNA or DNA, whereas yeast RNase H (35) can cleave RNA hybridized to DNA (24,68). Unlike a rad27⌬ strain, rhn35⌬ cells have a normal growth rate.…”

mentioning

confidence: 99%

Mutations in Yeast Replication Proteins That Increase CAG/CTG Expansions Also Increase Repeat Fragility

Callahan¹,

Andrews²,

Zakian

et al. 2003

Molecular and Cellular Biology

101

142

View full text Add to dashboard Cite

Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in laggingstrand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/ helicase Dna2, RNase HI, DNA ligase, polymerase ␦, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.

show abstract

Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII¹

Abstract: We cloned the Saccharomyces cerevisiae homologue of mammalian RNase HI, which itself is related to the prokaryotic RNase HII, an enzyme of unknown function and previously described as having minor activity in Escherichia coli.

Cited by 52 publications

References 30 publications

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

A Saccharomyces cerevisiae RNase H2 Interaction Network Functions To Suppress Genome Instability

Mutations in Yeast Replication Proteins That Increase CAG/CTG Expansions Also Increase Repeat Fragility

Contact Info

Product

Resources

About

Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII1

Abstract: We cloned the Saccharomyces cerevisiae homologue of mammalian RNase HI, which itself is related to the prokaryotic RNase HII, an enzyme of unknown function and previously described as having minor activity in Escherichia coli.

Cited by 52 publications

References 30 publications

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

A Saccharomyces cerevisiae RNase H2 Interaction Network Functions To Suppress Genome Instability

Mutations in Yeast Replication Proteins That Increase CAG/CTG Expansions Also Increase Repeat Fragility

Contact Info

Product

Resources

About

Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII¹