Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae

Abstract

Small looped mispairs are corrected by DNA mismatch repair. In addition, a distinct process called large loop repair (LLR) corrects heteroduplexes up to several hundred nucleotides in bacteria, yeast and human cells, and in cell-free extracts. Only some LLR protein components are known, however. Previous studies with neutralizing antibodies suggested a role for yeast DNA polymerase δ (Pol δ), RFC and PCNA in LLR repair synthesis. In the current study, biochemical fractionation studies identified FEN1 (Rad27) as another required LLR component. In the presence of purified FEN1, Pol δ, RFC and PCNA, repair occurred on heteroduplexes with loops ranging from 8 to 216 nt. Repair utilized a 5′ nick, with correction directed to the nicked strand, irrespective of which strand contained the loop. In contrast, repair of a G/T mismatch occurred at low levels, suggesting specificity of the reconstituted system for looped mispairs. The presence of RPA enhanced reactivity on some looped substrates, but RPA was not required for activity. Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5′ nick can be reconstituted in a purified system with FEN1 and Pol δ, together with PCNA and its loader RFC.

Factors affecting template switch recombination associated with restarted DNA replication

“…Ligation of the invading DNA strand to the nascent lagging strand of the oncoming fork would prevent further TS and consolidate the formation of a deletion, with branched DNA structures being resolved by nucleases (not shown) (Lorenz et al, 2009) or by the completion of DNA replication (Figure 7, steps 8a and 9a). In the latter case, the region being deleted would form a heterologous loop of ssDNA (Figure 7, step 9a), which could be removed by a large DNA loop repair pathway (Clikeman et al, 2001; Sommer et al, 2008).…”

Zap1‐dependent transcription from an alternative upstream promoter controls translation of RTC4 mRNA in zinc‐deficient Saccharomyces cerevisiae

“…It is worth noting that RTC4 LLT transcription passing through the adjacent GIS2 gene may also affect transcription of this downstream gene and we are investigating this hypothesis of dual gene regulation in ongoing experiments. RAD27 encodes a 5′‐to‐3′ DNA exonuclease that is important for Okazaki fragment removal during DNA replication and also acts in the repair of long heteroduplex DNA loops (Ayyagari et al, ; Sommer et al, ). Why Rad27 is repressed by Zap1 is also unclear.…”

Trinucleotide expansion in disease: why is there a length threshold?

“…Polymerase pausing is noted in long non-coding TNRs, and the size of the loops formed during fork reversal [ 18 ] or strand-switching [ 19 ] mechanisms have the potential to promote even larger loops. The endonucleases ( Table I ) that resolve the larger loops and their integration into genomic DNA are, as yet, unknown [ 20-38 ].…”