Interaction and stimulation of human FEN-1 nuclease activities by heterogeneous nuclear ribonucleoprotein A1 in alpha-segment processing during Okazaki fragment maturation.
High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.
Chai
Qing
Q
Division of Molecular Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010, USA.
Zheng
Li
L
Zhou
Mian
M
Turchi
John J
JJ
Shen
Binghui
B
D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D053938:DNA Fragmentation; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004789:Enzyme Activation; D045585:Flap Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C022262:Okazaki fragments; D010446:Peptide Fragments; C000619656:hnRNPA1 protein, human; D004247:DNA; D004256:DNA Polymerase I; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D005998:Glycine