PMC:4103454 / 4450-6491 JSONTXT

{"project":"2_test","denotations":[{"id":"24981866-20603015-20021734","span":{"begin":1194,"end":1198},"obj":"20603015"}],"text":"Processing of 5′ Flaps Is Conserved among FAN1 Orthologs\nOur initial aim was to crystallize and biochemically characterize FAN1, and to this end, we purified a range of FAN1 orthologs from human, mouse, and Pseudomonas (hFAN1, mFAN1, and pFAN1, respectively; Figure 1A), expressed in bacteria. To compare their nuclease activities, proteins were incubated with 5′ flap substrates labeled at either the 5′ (Figure 1B) or 3′ end (Figure 1C) of the flap strand. Figure 1B shows a similar cleavage pattern for hFAN1 and mFAN1, generating a long endonucleolytic product corresponding to cleavage 4 bp after the branchpoint and a short product (4 nt) corresponding to cleavage close to the 5′ end of the flap. This short product was the major band for hFAN1 but was less dominant for mFAN1. pFAN1 exhibited a different profile with strong cleavage 4 and 5 bp after the branchpoint but with no detectable cleavage at the 5′ end. However, all three enzymes displayed some 5′ to 3′ exonuclease activity following the initial cut (Figure 1C), suggesting that they differ in their ability to bind and/or cleave DNA ends, but not in their ability to process DNA branchpoints. As reported in MacKay et al. (2010), the FAN1 proteins do not have activity against Holliday junction (HJ) substrates (Figure S1A).\nThe apparent lack of pFAN1 activity at DNA ends suggested it as a good candidate for a more-detailed investigation of branchpoint processing. To this end, pFAN1 was incubated with 5′ flap substrates labeled at the 5′ end of either the flap strand (a) or the duplex strand (b) (Figures 1D and S1B). Whereas the a strand was cleaved almost to completion within the first minute, significant cleavage of the b strand only became apparent after 2 min, proceeding to completion in ∼40 min. The different kinetics of these two cleavage events suggests that the 5′ flap substrate is initially cleaved on the a strand and processed into a gapped duplex or a 5′ overhang, which is then targeted by another pFAN1 molecule in a slower reaction (Figure 1E)."}