Analysis of Human Flap Endonuclease 1 Mutants Reveals a Mechanism to Prevent Triplet Repeat Expansion

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109

206

The oxidized DNA base 8-oxoguanine (8-oxoG) is implicated in neuronal CAG repeat expansion associated with Huntington disease, yet it is unclear how such a DNA base lesion and its repair might cause the expansion. Here, we discovered size-limited expansion of CAG repeats during repair of 8-oxoG in a wildtype mouse cell extract. This expansion was deficient in extracts from cells lacking pol ␤ and HMGB1. We demonstrate that expansion is mediated through pol ␤ multinucleotide gap-filling DNA synthesis during long-patch base excision repair. Unexpectedly, FEN1 promotes expansion by facilitating ligation of hairpins formed by strand slippage. This alternate role of FEN1 and the polymerase ␤ (pol ␤) multinucleotide gap-filling synthesis is the result of uncoupling of the usual coordination between pol ␤ and FEN1. HMGB1 probably promotes expansion by stimulating APE1 and FEN1 in forming single strand breaks and ligatable nicks, respectively. This is the first report illustrating that disruption of pol ␤ and FEN1 coordination during long-patch BER results in CAG repeat expansion.

Section: Discussionmentioning

confidence: 99%

Coordination between Polymerase β and FEN1 Can Modulate CAG Repeat Expansion

Liu

Prasad

Beard

et al. 2009

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109

206

“…The yeast strains Kar212 (MAT␣, Kar1-1, ade trp1-289, ura3-52, leu2-3, 112) CTG-0, 85, and 155 were kindly provided by Catherine Freudenreich (Tufts University, Boston, MA). Assays for trinucleotide repeat stability and fragility were done as described by Liu and Bambara (37).…”

Section: Methodsmentioning

confidence: 99%

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

Singh

Zheng

Chavez

et al. 2007

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG) n and (GAA) n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG) n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for singlestranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

“…Initial cleavage by FEN1 was assayed on the standard substrate (lanes 5-8) and the RNA-containing substrate (lanes 18 -21) in the presence of PCNA/RFC and either pol ␦-wt or pol ␦ 3-01, as described under "Experimental Procedures." The extent of FEN1 cleavage into the downstream primer was assayed on the standard substrate (lanes 10 -13) and the RNA-containing substrate (lanes [23][24][25][26] in the presence of PCNA/RFC and either pol ␦-wt or pol ␦ 3-01, as described under "Experimental Procedures." Underlined bands indicate products corresponding to 10, 20, 30, or 40 nt.…”

Section: Fen1 Can Cleave Following Pol ␦-Wt and Pol ␦ 3-01 Displacemmentioning

confidence: 99%

Reconstituted Okazaki Fragment Processing Indicates Two Pathways of Primer Removal

Rossi

Bambara

2006

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Eukaryotic DNA replication proceeds discontinuously via Okazaki fragment synthesis and maturation on the lagging strand. DNA polymerase ␣ primes the lagging strand, synthesizing about 10 nucleotides (nt) 2 of RNA followed by an additional 10 -20 nt of DNA. The primer is extended by a complex of DNA polymerase ␦ (pol ␦), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC). Pol ␦ continues extension of the upstream fragment until it runs into the downstream fragment, displacing its 5Ј-end region into a flap. The flap can be cleaved by a nuclease to form a nick, which will be sealed by DNA ligase I to generate the continuous double-stranded DNA (1, 2).Cleavage of the flap can be completed by flap endonuclease 1 (FEN1), a 5Ј structure-specific endonuclease and the product of the RAD27 gene in Saccharomyces cerevisiae (1). One proposed model suggests FEN1 cleavage of primarily short flaps displaced by pol ␦ (3-5). As pol ␦ displaces the downstream primer into a flap, FEN1 can track from the 5Ј-end (6, 7) to the base of the flap for cleavage (6,8,9) that generates a nick for ligation. Through Okazaki fragment processing reconstitution studies in vitro, Ayyagari et al. (3) showed that short flaps, 10 nt in length, are processed efficiently by FEN1 to generate ligatable replication intermediates. More recent experimentation revealed that in the presence of pol ␦ and FEN1, strand-displacement and cleavage generated mainly mononucleotide products (5). In contrast, strand-displacement by mutant forms of pol ␦, deficient in its proofreading 3Ј-5Ј exonuclease activity, and cleavage by FEN1 generated mono-through hexanucleotide products (5). It was proposed that the 3Ј-5Ј exonuclease activity of pol ␦, coordinated with FEN1, prevents extensive strand-displacement because pol ␦ 3Ј-5Ј exonuclease mutants exhibit greater stranddisplacement activity than wild-type pol ␦ (4, 5). This idea is supported by the synthetic lethal interaction between 3Ј-5Ј exonuclease mutants of pol ␦ (pol3-exo) and either rad27 deletion mutants or those that are compromised for interaction with its cofactor PCNA (rad27-p) (10 -12). In addition, the rare pol3-exo rad27-p double mutants that are not lethal require intact recombination and double-strand break repair machinery (12). Altogether, these results suggest that with wild-type proteins, the system is generally designed to strand-displace and cleave short flaps, while failure to cleave at the short stage, either because strand-displacement is enhanced or nuclease cleavage inhibited, leads to the possibility that long flaps occasionally form (3-5).If a flap escapes cleavage by FEN1, it can become long and follow a secondary pathway. A model put forward by Bae et al. (13) asserts that flaps greater than 27 nt in length are coated by the single-stranded-binding protein replication protein A (RPA). RPA inhibits cleavage by FEN1 but stimulates cleavage by Dna2 nuclease/helicase (13,14). Dna2, like FEN1, must track from the 5Ј-end of the flap to...