Abstract
DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3′-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3′-flap. This recognition event allosterically signals hydrolytic removal of the 5′-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved ‘wedge’ residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated ‘loop–wedge’ mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3′-flap verification to function.
Highlights
Structure-selective nucleases (SSNs) carry out many important functions in DNA replication and repair, and their actions are essential in maintaining genome stability
Strand displacement synthesis during DNA replication (Figure 1A) produces intermediate structures with protruding overhangs, or flaps, which must be removed before the synthesis of new DNA can be completed by ligation
This task is carried out by flap endonuclease 1 (FEN1), a representative and well-studied SSN, which recognizes its target DNA junction structures with high selectivity [1]. This role in replication is the primary cellular function of FEN1, it participates in DNA repair processes that employ strand displacement synthesis––including long-patch base excision repair (LP-BER) [2] and ribonucleotide excision repair (RER) [3]––and is rapidly recruited to sites of DNA damage in vivo [4]
Summary
Structure-selective nucleases (SSNs) carry out many important functions in DNA replication and repair, and their actions are essential in maintaining genome stability. Strand displacement synthesis during DNA replication (Figure 1A) produces intermediate structures with protruding overhangs, or flaps, which must be removed before the synthesis of new DNA can be completed by ligation This task is carried out by flap endonuclease 1 (FEN1), a representative and well-studied SSN, which recognizes its target DNA junction structures with high selectivity [1]. This role in replication is the primary cellular function of FEN1, it participates in DNA repair processes that employ strand displacement synthesis––including long-patch base excision repair (LP-BER) [2] and ribonucleotide excision repair (RER) [3]––and is rapidly recruited to sites of DNA damage in vivo [4]. 3 -flap recognition is essential for the efficiency of DNA replication and repair involving FEN1
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