Human replication protein A melts a DNA triple helix structure in a potent and specific manner.
Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, to destabilize DNA triplexes and tetraplexes. Biochemical studies demonstrate that RPA efficiently melts an intermolecular DNA triple helix consisting of a pyrimidine motif third strand annealed to a 4 kb duplex DNA fragment at protein concentrations equimolar to the triplex substrate. Heterologous single-stranded DNA binding proteins ( Escherichia coli SSB, T4 gene 32) melt the triplex substrate very poorly or not at all, suggesting that the triplex destabilizing effect of RPA is specific. In contrast to the robust activity on DNA triplexes, RPA does not melt intermolecular G4 tetraplex structures. Cellular assays demonstrated increased triplex DNA content when RPA is transiently repressed, suggesting that RPA melting of triple helical structures is physiologically important. On the basis of our results, we suggest that the abundance of RPA known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form from human genomic DNA sequences.
Wu
Yuliang
Y
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Rawtani
Nina
N
Thazhathveetil
Arun Kalliat
AK
Kenny
Mark K
MK
Seidman
Michael M
MM
Brosh
Robert M
RM
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D004277:DNA, Single-Stranded; D051716:Replication Protein A; C051802:triplex DNA; D004247:DNA