On the molecular weight and subunit composition of calf thymus ribonuclease H1

Rong, Yi; Carl, Philip L.

doi:10.1021/bi00454a012

Cited by 43 publications

(25 citation statements)

References 28 publications

(40 reference statements)

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“…They are also coincident with protein bands at 70, 30, 27, and 18 kDa. This represents nearly homogeneous RNase HI where the lower molecular weight species are degradation products of the full-length 70-kDa protein (7). RNase HI was also purified by a second method that involved different types of fractionation from the first procedure.…”

Section: Resultsmentioning

confidence: 99%

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Junction ribonuclease: An activity in Okazaki fragment processing

Murante

Henricksen

Bambara

1998

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

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The initiator RNAs of mammalian Okazaki fragments are thought to be removed by RNase HI and the 5-3 f lap endonuclease (FEN1). Earlier evidence indicated that the cleavage site of RNase HI is 5 of the last ribonucleotide at the RNA-DNA junction on an Okazaki substrate. In current work, highly purified calf RNase HI makes this exact cleavage in Okazaki fragments containing mismatches that distort the hybrid structure of the heteroduplex. Furthermore, even fully unannealed Okazaki fragments were cleaved. Clearly, the enzyme recognizes the transition from RNA to DNA on a single-stranded substrate and not the RNA͞DNA heteroduplex structure. We have named this junction RNase activity. This activity exactly comigrates with RNase HI activity during purification strongly suggesting that both activities reside in the same enzyme. After junction cleavage, FEN1 removes the remaining ribonucleotide. Because FEN1 prefers a substrate with a single-stranded 5-f lap structure, the single-stranded activity of junction RNase suggests that Okazaki fragments are displaced to form a 5-tail prior to cleavage by both nucleases. RNase HI is involved in the removal of the initiator RNA primers of Okazaki fragments, an essential step during chromosomal DNA replication of the lagging strand (10). These RNAs, 7-14 nucleotides in length, are generated by the primase activity of DNA polymerase ␣ (pol ␣) and prime DNA synthesis of both the leading and lagging strands at the DNA replication fork. The pol activity of this same enzyme then extends each primer with deoxynucleotides. Pol ␣ is thought to be replaced by pol ␦ that continues synthesis to the next downstream fragment. There is considerable evidence that the RNA is removed by the combined action of RNase HI and a 5Ј to 3Ј exo͞endonuclease (11-13). This latter enzyme has been called flap endonuclease (FEN1) (14) in mammals and is known as RAD two homolog (RTH1) and RAD27 in Saccharomyces cerevisiae (15). Removal of the RNA is required before DNA synthesis and ligation can complete the replication process.The exact functions of RNase HI in DNA replication have been the subject of several studies. Drosophila RNase HI was found to stimulate synthesis by pol ␣ (4). In addition, RNases H from yeast and calf thymus (3, 6) associate with and stimulate their cognate pol ␣. These observations suggest that RNase HI and pol ␣ form a complex in vivo capable of synthesis and removal of RNA primers. Reconstitution assays with both mouse cell and SV40 replication proteins require RNase HI for the maturation of lagging strands in vitro (12, 13).Although cleavage of long RNA͞DNA hybrids is random, cleavage of certain substrates is clearly structure specific. Eder et al. (16) found that when a series of ribonucleotides were followed by a 3Ј DNA segment and annealed to DNA to form a duplex, human RNase HI preferentially cleaved to leave a DNA segment with a 5Ј monoribonucleotide. In reactions reconstituting Okazaki fragment processing, RNase HI from calf thymus similarly cleaved the RNA-DNA stra...

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Section: Resultsmentioning

confidence: 99%

“…Two classes of RNase H, RNase HI and II, have been identified in a variety of eukaryotes, purified and characterized (1)(2)(3)(4)(5)(6)(7)(8) as a cofactor. Both forms of RNase H randomly degrade RNA in long RNA͞DNA hybrid structures (2,9).…”

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confidence: 99%

Junction ribonuclease: An activity in Okazaki fragment processing

Murante

Henricksen

Bambara

1998

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

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“…RNase H enzymes function as endonucleases requiring divalent cations (e.g., Mg 2ϩ , Mn 2ϩ ) to produce cleavage products with 5Ј-phosphate and 3Ј-hydroxyl termini (Crouch and Dirksen, 1982). RNase H activity seems to be ubiquitous in eukaryotes and bacteria (Busen, 1980;Rong and Carl, 1990;Itaya and Kondo, 1991;Kanaya and Itaya, 1992;Eder et al, 1993). Two classes of RNase H enzymes have been identified in mammalian cells (Busen, 1980;Masutani et al, 1990;Eder et al, 1993;Frank et al, 1994;Wu et al, 1998).…”

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confidence: 99%

Human RNase H1 Discriminates between Subtle Variations in the Structure of the Heteroduplex Substrate

Lima¹,

Rose²,

Nichols³

et al. 2006

Mol Pharmacol

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In a previous study, we demonstrated that the sugar conformation and helical geometry of the heteroduplex substrate at the catalytic site of human RNase H1 directs the selective recognition of the substrate by the enzyme (J Biol Chem 279: [36317][36318][36319][36320][36321][36322][36323][36324][36325][36326] 2004). In this study, we systematically introduced 2Ј-methoxyethoxy (MOE) nucleotides into the antisense oligodeoxyribonucleotide (ASO) of the heteroduplex to alter the helical geometry of the substrate. The MOE substitutions at the 3Ј and 5Ј poles of the ASO resulted in fewer cleavage sites and slower cleavage rates compared with the unmodified substrates. Furthermore, a greater reduction in cleavage activity was observed for MOE substitutions at the 5Ј pole of the ASO. The 3Ј-and 5Ј-most cleavage sites were positioned two and four to five base pairs, respectively, from the nearest MOE residues, suggesting a conformational transmission of the MOE/RNA helical geometry into the DNA/RNA portion of the heteroduplex. Similar conformational transmission was observed for Okazaki-like substrates containing deoxyribonucleotide substitutions at the 3Ј pole of the oligoribonucleotide. Finally, the heteroduplex substrates exhibited preferred cleavage sites that were cleaved 2-to 3-fold faster than other sites in the substrate, and these sites exhibited the greatest influence on the initial cleavage rates. The data presented here offer further insights into the role substrate structure plays in directing human RNase H1 activity as well as the design of effective ASOs.

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“…RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2)(3)(4)(5)(6)(7). Although RNases H constitute a family of proteins of varying molecular mass, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes.…”

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confidence: 99%