Dna2 dating

The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.

encodes a 172 k Da protein that is a multifunctional enzyme and thus has been implicated in several different aspects of DNA transactions (1–6).

All oligonucleotides used to construct various DNA substrates were synthesized commercially (Genotech, Taejeon, Korea) and gel-purified prior to use.

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Immunoaffinity-purified Dna2 fusion protein from crude extracts displayed single-stranded (ss)DNA-dependent ATPase and DNA helicase activities (2,7).

Recombinant Dna2 isolated from insect cells was shown to possess intrinsic ss DNA-specific endonuclease activity in addition to helicase activity (7).

These included six histidines (bold) and the Xpress epitope (underlined) fused to its N-terminal methionine to facilitate detection and purification of the recombinant Dna2 proteins.

HX-Dna2N behaved similarly to HX-Dna2 in chromatographic steps and was purified using the same procedure described for HX-Dna2 (7).

Overexpression of full-length Dna2 caused arrest of cell growth (14), an effect that is not observed when a truncated Dna2 protein lacking the N-terminal 105 amino acids was overexpressed (11).

Moreover, overexpression of an N-terminal portion of Dna2 causes significant derepression of a gene located near a telomere in yeast (15), indicating that the N-terminal part of Dna2 may play a unique or regulatory role in DNA transactions.

A DNA substrate used to examine endonuclease activity of the Dna2 proteins was prepared by hybridizing a 73mer to ΦX174 ssc DNA as described (7).

The substrate contains a free 21 nt ss DNA 5′-tail connected to a 52 bp duplex.

These enzymatic properties of Dna2 provided a biochemical basis for a role in Okazaki fragment maturation (1).

Consistent with these (DNA ligase I), all of which are required for Okazaki fragment maturation (5).

Recombinant Dna2 proteins including HX-Dna2 (wild-type) and HX-Dna2Δ405N (lacking the N-terminal 405 amino acids) were expressed in insect cells and purified as described (7).

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