Creation and Removal of Embedded Ribonucleotides in Chromosomal DNA during Mammalian Okazaki Fragment Processing

Rumbaugh, Jeffrey A.; Murante, Richard S.; Shi, Shuhui; Bambara, Robert A.

doi:10.1074/jbc.272.36.22591

Cited by 51 publications

(42 citation statements)

References 49 publications

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“…Results of the mismatch experiment show that recognition of the junction cleavage site by RNase HI does not require the helical structure present at a normal RNA-DNA junction in a heteroduplex. While surprising, this conclusion is consistent with the observation that RNase HI cleaves 5Ј of a single ribonucleotide embedded in a DNA-RNA-DNA͞DNA heteroduplex (16,24), anticipated to have a helical structure different from that of a duplex Okazaki fragment substrate. This observation also explains why initiator RNA mismatches do not disrupt mammalian DNA replication.…”

Section: Resultssupporting

confidence: 72%

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: Resultssupporting

confidence: 72%

Junction ribonuclease: An activity in Okazaki fragment processing

Murante

Henricksen

Bambara

1998

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

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“…Absence of the upstream primer inhibits the ability of FEN1 to remove exonucleolytically the 5Ј-monoribonucleotide that remains after RNase H degradation of the RNA (31,32). This suggests that strand-displacement synthesis stimulates FEN1-directed removal of the initiator RNA.…”

mentioning

confidence: 94%

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

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139

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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...

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“…Moreover, they have been identified as the sole enzymes able to recognise and cleave a single ribonucleotide embedded in dsDNA (Eder and Walder, 1991), thereby contrasting to type 1 RNases H that requires at least four ribonucleotides for cleavage (Ohtani et al, 1999a). As mentioned earlier, single ribonucleotides embedded in dsDNA can arise from external damaging agents (Von Sonntag and Schulte-Frohlinde, 1978), and can occur by intrinsic RNA ligation (Rumbaugh et al, 1997) or erroneous nucleotide incorporation during DNA replication (Nick McElhinny et al, 2010a;Nick McElhinny et al, 2010b). The presence of riboses in DNA has been shown to induce a helical alteration, promoting a B-to A-form transition in DNA (Horton and Finzel, 1996).…”

Section: Physiological Roles For Rnases Hii/2mentioning

confidence: 99%

“…Likewise, defect of the catalytic mutant MmuRNase H2 to hydrolyse single embedded ribonucleotides pointed toward a role for eukaryotic RNase H2 in DNA repair (Shaban et al, 2010). Furthermore, based on genetic and biochemical results, removal of single embedded ribonucleotides seems to involve at least type 2 RNases H, Fen1 (Flap Endonuclease 1) and PCNA (Bubeck et al, 2011;Meslet-Cladiere et al, 2007;Nick McElhinny et al, 2010a;Rumbaugh et al, 1997;Rydberg and Game, 2002), with PCNA:RNase HII/2 complex acting as a sensor of erroneous ribonucleotides. The association of such protein components likely suggests a role of type 2 RNases H in base excision repair (BER) to accomplish the removal of mutagenic ribonucleotides.…”

Section: Physiological Roles For Rnases Hii/2mentioning

confidence: 99%

“…Cleavage specificities for substrates containing single or few ribonucleotides embedded in double-stranded DNA (dsDNA) of type 2 RNases H are now well documented. Such structural substrates can arise in vivo during Okazaki fragment processing from intrinsic RNA ligation activity (Rumbaugh et al, 1997) or erroneous nucleotide incorporation by DNA polymerases (Nick McElhinny et al, 2010a;Nick McElhinny et al, 2010b), and during exposure to external damaging agents (Von Sonntag and Schulte-Frohlinde, 1978). Initial studies revealed that the archaeal TkoRNase HII was active on four ribonucleotides embedded in dsDNA (DNA-RNA 4 -DNA/DNA).…”

Section: Introductionmentioning

confidence: 99%

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