DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D.
Nucleic Acids Res
; 38(4): 1149-62, 2010 Mar.
Article
em En
| MEDLINE
| ID: mdl-19969545
ABSTRACT
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 delta and alpha, 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 delta 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 delta 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.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Sítios Frágeis do Cromossomo
/
DNA Polimerase III
/
Replicação do DNA
/
RecQ Helicases
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos