Single-Molecule Studies of the Effects of Nucleoid-Associated Proteins On DNA Helicase Activity

Leah Katz

Laser beam system for optical trap.

Laser beam system for optical trap.

Undergraduate Student Project


Nucleoid-Association Proteins (NAPs) are "histone-like" proteins that compact DNA into the nucleoid in E.coli. NAPs compact DNA by bending, bridging, and wrapping DNA. In addition, NAPs have control over transcription, DNA repair, replication, and recombination. In order for DNA repair, replication, and recombination to occur, double-stranded DNA needs to be unwound into single-stranded DNAs. RecBCD is a DNA helicase that unwinds double-stranded DNAs. RecBCD is an important enzyme for the initiation of double-stranded DNA break repair (homologous recombination). My research seeks to determine the effects NAPs have on the RecBCD enzyme, which provides greater insight into how NAPs affect homologous recombination.


Nucleoid-Associated Proteins (NAPs) contribute to the dynamic organization of the bacterial nucleoid and control DNA transactions.  While the role of NAPs in controlling gene expression is well known their potential roles in genetic recombination and DNA repair are less well understood.  To understand how NAPs affect recombination, we have studied the effects of the predominant NAPs on DNA unwinding by the recombination initiator RecBCD. First, using a bulk-phase assay the effects on DNA unwinding were assessed.  Second, a single DNA molecule approach is used.  This approach combines optical tweezers with video-fluorescence microscopy and microfluidics to visualize DNA transactions in real-time.  We observed the binding of NAPs to single, optically-trapped bacteriophage lambda DNA molecules in real-time. Finally, the effects of DNA bound-NAPS on DNA unwinding by the RecBCD DNA helicase were studied in real-time. A model will be presented to explain the importance of these findings.

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