Proliferating cell nuclear antigen originally identified as an antigen that is expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle. Part of the protein was sequenced and that sequence was used to allow isolation of a cDNA clone. PCNA helps hold DNA polymerase epsilon (Pol ε) to DNA. PCNA is clamped to DNA through the action of replication factor C (RFC), which is a heteropentameric member of the AAA+ class of ATPases. Expression of PCNA is under the control of E2F transcription factor-containing complexes. Since DNA polymerase epsilon is involved in resynthesis of excised damaged DNA strands during
Single-cell DNA template strand sequencing cells are treated with bromodeoxyuridine (BrdU) which is then incorporated into their nascent DNA, acting as a substitute for thymidine. After at least one replication event has occurred, the daughter cells are synchronized at the G2 phase and individually separated by fluorescence-activated cell sorting (FACS). The cells are directly sorted into lysis buffer and their DNA is extracted. Having been arrested at a specified number of generations (usually one), the inheritance patterns of sister chromatids can be assessed. The following methods concentrate on the DNA sequencing of a single daughter cell’s DNA. At this point the chromosomes are composed of