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The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each(More)
Determining numbers of proteins bound to large DNAs is important for understanding their chromosomal functions. Protein numbers may be affected by physical factors such as mechanical forces generated in DNA, e.g. by transcription or replication. We performed single-DNA stretching experiments with bacterial nucleoid proteins HU and Fis, verifying that the(More)
The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most(More)
HU is one of the most abundant proteins in bacterial chromosomes and participates in nucleoid compaction and gene regulation. We report experiments using DNA stretching that study the dependence of DNA condensation by HU on force, salt and HU concentration. Previous experiments at sub-physiological salt levels revealed that low concentrations of HU could(More)
The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis-DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not(More)
Most site-specific recombinases can be grouped into two mechanistically distinct families. Whereas tyrosine recombinases exchange DNA strands through a Holliday intermediate, serine recombinases such as Hin generate double-strand breaks in each recombining partner. Here, site-directed protein crosslinking is used to elucidate the configuration of protein(More)
BACKGROUND Hin is a member of an extended family of site-specific recombinases--the DNA invertase/resolvase family--that catalyze inversion or deletion of DNA. DNA inversion by Hin occurs between two recombination sites and requires the regulatory protein Fis, which associates with a cis-acting recombinational enhancer sequence. Hin recombinase dimers bind(More)
Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the(More)
Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate(More)
Replication and transcription activator (RTA), an immediate-early gene product of gamma-2 herpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gamma herpesvirus 68 (MHV-68), plays a critical role in controlling the viral life cycle. RTA acts as a strong transcription activator for several downstream genes of KSHV and MHV-68(More)