FEN1-mediated α-segment error editing during Okazaki fragment maturation

Abstract

DNA-replication-repair machinery determines the non-random occurrence of variations across the genome. During eukaryotic nuclear-genome replication, most parts of the leading and lagging strand are synthesized by high fidelity DNA polymerase ϵ and δ respectively. However, a third enzyme polymerase (Pol) α-primase initiates each synthesis event. During Okazaki fragment, down-stream of the RNA primer and synthesis, DNA fragment synthesized by proofreading-deficient Pol-α, which we termed as α-segment, provide major source of mutations and undergo DNA repair process to remove the incorporated errors.1 The α-segment contributes ∼1.5% of total genome. A series of the very recent papers made available by Nature press indicates that despite Okazaki fragment processing, DNA synthesized by Pol α is retained in vivo.2-5 DNA-binding proteins including histones and transcription factors that rapidly re-associate, post-replication, to act as partial barriers to Pol-δ-mediated displacement of the α-segment, resulting increased mutation rates in the region. What is the cellular mechanism that minimizes the mutation frequency in the alpha-segment? Limited evidence is available. Pol α variant strains have mild mutator phenotypes, resulting from DNA replication errors generated by Pol α, and their mutation rates are synergistically increased by loss of MSH2-dependent mismatch repair (MMR). Thus, MSH2-dependent MMR in yeast plays a major role in correcting mismatches in the α-segment.1 Additional study indicated that some mismatches generated by yeast Pol α are excised by exonuclease 1 (EXO1), a 5′ exonuclease involved in MMR.6 Interestingly, the loss of MMR resulting from deleting EXO1 is mild compared to the loss of MMR from deleting MSH2, implying the existence of a MSH2-dependent but EXO1-independent mechanism that removes mismatches from the α-segment. In our paper currently in press with EMBO Journal,7 we investigated the molecular mechanism mediated by FEN1 and MSH2 functional complex to edit the errors in the α-segment. Our unique substrate designing strategy segregates the RNA primer removal (RPR) and α-segment error editing (AEE) activities of FEN1 and characterize efficiency of functional enzymatic component of nuclear extract (NEs). Interestingly, besides its well-characterized RNA primer removl function, FEN1 was able to go on in an exonuclease fashion in presence of the mis-paired nucleotides in the α-segment and remove them within a limited distance (12 nts) from the nick MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5′ ends of DNA substrates. Mouse cells and mice encoding FEN1 AEE defective mutations display a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility.7 The proposed AEE mechanism is significant because it effectively removes replication errors while concomitantly maturing Okazaki fragments, which is a rate-limiting step in DNA replication.7 However, if a mutation occurs in AEE components that deregulates the process and leads to constitutive editing and futile cycles, it could affect DNA ligation and result in spontaneous DNA strand breaks and consequently, various forms of chromosome aberrations. Such a mutation would be different from the ones due to defects in RPR process, of which the major form is the duplication mutation. Both the mutations that result in DNA strand breaks and aneuploidy and the duplication mutation, are hallmarks of human cancer. The current study identifies a novel role of FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.