An assay system for factors involved in mammalian DNA replication

Evidence for the involvement of single‐strand DNA‐binding proteins in mammalian DNA replication

“…In this communication, an experimental system that may contribute to the elucidation of the functions of mammalian ssDNA-binding proteins willbe presented. As previously reported [6], cell extract (i.e. a supernatant obtained by high speed centrifugation of homogenized CHO cells) stimulates semiconservative DNA replication in partially lysed CHO cells.…”

“…Herring sperm DNA was purchased from Fluka, and [14C]DNA was purified from CHO cells as described [6]. Metrizamide (analytical grade) was obtained from Nyegaard; Co A/S and DNase I from Worthington Biochemical.…”

“…Metrizamide (analytical grade) was obtained from Nyegaard; Co A/S and DNase I from Worthington Biochemical. The source of other compounds has been previously indicated [6].…”

“…Exponentially growing CHO cells were prelabeled with [14C]dThd (2 nCi/ml; 50 Ci/mol) and detached from petri dishes by trypsin treatment. Partially lysed CHO cells were prepared by treatment with a buffer containing 0.1% Brij-58 and 240 mM KC1 as previously described and as referred to as "highly lysed cells" [6]. In this preparation, 20-50% of nuclei were essentially free of cytoplasm and SO-80% of nuclei were surrounded with little cytoplasm.…”

“…To correct for differences in DNA synthesis due to slight variations of cell numbers in the test tubes, 3H/14C ratios were calculated. Further details of this assay system have been described previously [6].…”