Dynamic Removal of Replication Protein A by Dna2 Facilitates Primer Cleavage during Okazaki Fragment Processing in Saccharomyces cerevisiae

Stewart, Jason A.; Miller, Adam S.; Campbell, Judith L.; Bambara, Robert A.

doi:10.1074/jbc.m805965200

Cited by 44 publications

(74 citation statements)

References 48 publications

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“…When RPA was also included in the reactions (lanes 10 -13), the distribution shifted to shorter products. This is consistent with results showing that RPA stimulates Dna2 cleavage efficiency, such that the Dna2 cleaved sooner as it tracked from the 5Ј-end of the flap (21). As a control, the reactions with Pif1 were repeated in the absence of pol ␦.…”

Section: Resultssupporting

confidence: 89%

“…As expected, RPA inhibited FEN1 cleavage (lane 4) ϳ50%. Dna2 was also able to cleave the substrate (lane 5), and RPA stimulated Dna2 to cleave past the base of the flap (lane 6), as previously reported (21). The combination of FEN1 and Dna2 resulted in more cleavage than either nuclease alone (lane 7), also expected because Dna2 stimulates FEN1 (16).…”

Section: Dna2 Rescues Rpa Inhibition Of the Final Ligation Product-supporting

confidence: 78%

“…RPA binding to a flap has been shown to stimulate Dna2 cleavage (16). Therefore, if a flap becomes long enough to bind RPA, Dna2 binds and cleaves it to a length of 5-10 nucleotides from which RPA dissociates (21). FEN1 can then enter the flap, displace the Dna2, and then cleave at the base to make the nick for ligation (16,18,22).…”

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confidence: 99%

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Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

Pike

Burgers

Campbell

et al. 2009

Journal of Biological Chemistry

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We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase ␦ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase ␦ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5Ј to 3Ј direction. A leading strand is synthesized by DNA polymerase ⑀ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase ␦ (pol ␦) 3 in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ϳ150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase ␣/primase (pol ␣) initiates each fragment by synthesizing an RNA/DNA primer consisting of ϳ1-nt of RNA and ϳ10 -20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol ␦ then complexes with PCNA and extends the primer. When pol ␦ reaches the 5Ј-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol ␦ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7-9). FEN1 binds at the 5Ј-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol ␦ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol ␦ and FEN1 also tends to keep flaps...

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Section: Resultssupporting

confidence: 89%

Section: Dna2 Rescues Rpa Inhibition Of the Final Ligation Product-supporting

confidence: 78%

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Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

Pike

Burgers

Campbell

et al. 2009

Journal of Biological Chemistry

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“…The multiple levels of regulation of these proteins, involving nuclear import/export and their expression/degradation in the same signaling complexes and pathways, appear to ensure the timely onset of S phase or anaphase. Of the other identified proteins, Dna2 has been reported to function as a nuclease/helicase that is involved in Okazaki fragment processing in replication and in the repair of DNA double-strand breaks via homologous recombination (31,32), and Psy4 is involved in the repair of cross-linked DNA (33), indicating a tight coupling of DNA repair to cell cycle control. Dna2 resects DNA double-strand-break ends in concert with other helicase and nucleases including Sae2 (34), which is regulated by CDK1 (35).…”

Section: Discussionmentioning

confidence: 99%

Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

Kosugi

Hasebe

Tomita

et al. 2009

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

1,109

1,028

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The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-␣-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-␣/␤ pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1-and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.CDK ͉ computational method ͉ nuclear localization signal ͉ nuclear import ͉ phosphorylation R egulated nuclear import controls the nuclear function of many proteins that shuttle between the nucleus and cytoplasm or other cellular compartments in response to certain physiological conditions or extracellular stimuli (1). Nucleocytoplasmic shuttling comprises both nuclear import and nuclear export activities. Nuclear import is mediated by the interaction of nuclear localization signals (NLSs) with transport receptor importins, among which the importin-␣ family recognizes most major classes of NLSs with 1 or 2 basic stretches, called the classical NLSs (2-4). Six different classes of monopartite and bipartite NLSs bind to distinct binding grooves of importin-␣ (5-7). Nuclear export is mediated by nuclear export signals (NESs), which are recognized by Crm1/exportin or Msn5 in yeast (1, 8). The regulation of these 2 antagonistic activities directs a protein to either the nucleus or the cytoplasm. In general, these activities are regulated by the modification of the NLSs/NESs, by intermolecular or intramolecular interactions that lead to the structural masking of NLSs/NESs, or by the supply of an NLS/NES by another protein (9).Phosphorylation within or around an NLS is the usual strategy for the regulated nuclear transport of a protein (4, 10-13). It has been shown that acidic amino acids adjacent to the basic core ...

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“…cerevisiae Dna2 can dissociate the RPA from a long flap (Bae et al 2001;Stewart et al 2008). Using a mechanism very similar to that of FEN1, Dna2 binds the flap base, and then threads the free 5 0 end of the flap ).…”

Section: Long Flap Pathwaymentioning

confidence: 99%

Okazaki Fragment Metabolism

Balakrishnan

Bambara

2013

Cold Spring Harbor Perspectives in Biology

138

109

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Cellular DNA replication requires efficient copying of the double-stranded chromosomal DNA. The leading strand is elongated continuously in the direction of fork opening, whereas the lagging strand is made discontinuously in the opposite direction. The lagging strand needs to be processed to form a functional DNA segment. Genetic analyses and reconstitution experiments identified proteins and multiple pathways responsible for maturation of the lagging strand. In both prokaryotes and eukaryotes the lagging-strand fragments are initiated by RNA primers, which are removed by a joining mechanism involving strand displacement of the primer into a flap, flap removal, and then ligation. Although the prokaryotic fragments are 1200 nucleotides long, the eukaryotic fragments are much shorter, with lengths determined by nucleosome periodicity. The prokaryotic joining mechanism is simple and efficient. The eukaryotic maturation mechanism involves many enzymes, possibly three pathways, and regulation that can shift from high efficiency to high fidelity.

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Dynamic Removal of Replication Protein A by Dna2 Facilitates Primer Cleavage during Okazaki Fragment Processing in Saccharomyces cerevisiae

Cited by 44 publications

References 48 publications

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

Okazaki Fragment Metabolism

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