Replication forks are associated with the nuclear matrix.
It has been proposed that DNA in eukaryotic cells is synthesized via replication complexes that are fixed to a proteinaceous nuclear matrix. This model has not been universally accepted because the matrix and its associated DNA are usually prepared under hypertonic conditions that could facilitate non-specific aggregation of macromolecules. We therefore investigated whether different ionic conditions can significantly affect the association of nascent DNA with the nuclear matrix in cultured mammalian cells. Matrices were prepared either by a high salt method or by hypotonic or isotonic LIS extraction. Chromosomal DNA was subsequently removed by digestion with either DNAse I or EcoRI. With all methods of preparation, we found that newly synthesized DNA preferentially partitioned with the nuclear matrix. Furthermore, when the matrix-attached DNA fraction was analyzed by two-dimensional gel electrophoresis, we found that it was markedly enriched for replication forks. We therefore conclude that attachment of DNA to the matrix in the vicinity of replication forks is not induced by conditions of high ionic strength, and that replication may, indeed, occur on or near the skeletal framework provided by the nuclear matrix. From a practical standpoint, our findings suggest a strategy for greatly increasing the sensitivity of two important new gel electrophoretic methods for the direct mapping of replication fork movement through defined chromosomal domains in mammalian cells.
Vaughn
J P
JP
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Dijkwel
P A
PA
Mullenders
L H
LH
Hamlin
J L
JL
D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015530:Nuclear Matrix / Q000032:analysis / Q000378:metabolism*
D004247:DNA; D015246:Deoxyribonuclease EcoRI; D003850:Deoxyribonuclease I