The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells.
Primer recognition proteins (PRP) enable DNA polymerase alpha to utilize efficiently DNA substrates with low primer to template ratios. We have previously identified the protein-tyrosine kinase substrate annexin II, and the glycolytic enzyme 3-phosphoglycerate kinase as components of PRP. As a step towards elucidation of the role of PRP in the process of DNA replication, we have investigated the subcellular distribution and specific association of these proteins with the nuclear matrix in HeLa cells. Nuclear extracts prepared from HeLa cells in S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2 inhibitory activity of annexin II. Monomer annexin II is approximately equally distributed between the nuclear and cytoplasmic fractions, while a majority of PGK is in the cytoplasm. Immunoblot analyses reveal the presence of these two proteins in nuclei, specifically associated with the nuclear matrix. This is further confirmed by observation of the presence of annexin II and PGK in isolated nuclear matrices by immunoelectron microscopy. The phospholipase A2 inhibitory activity of annexin II colocalizes with the nuclear matrix-bound annexin II. A related protein, annexin I, is not detectable in the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin II is found to be associated with the nuclear matrix. Attempts to dissociate PGK and annexin II from the nuclear matrix with octyl-beta-glucoside, high salt or metal ion chelators were unsuccessful, suggesting that the interaction is very strong.
Vishwanatha
J K
JK
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198-4525.
Jindal
H K
HK
Davis
R G
RG
D017302:Annexins; D002135:Calcium-Binding Proteins / Q000032:analysis*; D003600:Cytosol / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D015530:Nuclear Matrix / Q000737:chemistry* / Q000648:ultrastructure; D010735:Phosphoglycerate Kinase / Q000032:analysis*
D017302:Annexins; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D010735:Phosphoglycerate Kinase