DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

• Published in: Nucleic acids research 2010-03, Vol.38 (4), p.1149-1162
• Main Authors: Shah, Sandeep N, Opresko, Patricia L, Meng, Xiao, Lee, Marietta Y.W.T, Eckert, Kristin A
• Format: Article
• Language: English
• Subjects: Base Sequence; Chromosome Fragile Sites; DNA - biosynthesis; DNA - chemistry; DNA Polymerase III - metabolism; DNA Replication; Exodeoxyribonucleases - metabolism; Genome Integrity, Repair and; HeLa Cells; Humans; RecQ Helicases - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkp1131

Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases δ and α, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol δ was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]₂₄ and [A/T]₁₉₋₂₈, compared with a control region with minimal secondary structure. Pol δ processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors--density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.