Boston, Mass. — North­eastern Uni­ver­sity sci­en­tists have dis­cov­ered a new and unique DNA binding prop­erty of a pro­tein in E. coli. Penny J. Beuning, Assis­tant Pro­fessor in the Depart­ment of Chem­istry and Chem­ical Biology, spent the last two years researching double and single-​​stranded DNA binding of E. coli DNA poly­merase III alpha pro­tein and notes that her find­ings have poten­tial for devel­oping a new antibac­te­rial target. Beuning’s results have recently been pub­lished in ACS Chem­ical Biology in an article titled “Dis­tinct Double– and Single-​​Stranded DNA Binding of E. coli Replica­tive DNA Poly­merase III Alpha Subunit”.

This work rep­re­sents the col­lab­o­ra­tive effort of the North­eastern lab­o­ra­to­ries of Beuning and Mark C. Williams, Asso­ciate Pro­fessor of Physics, and involved researchers Micah J. McCauley, Leila Shokri, and Jana Sef­cikova from both lab­o­ra­to­ries. Addi­tion­ally, Česlovas Ven­clovas, of the Insti­tute of Biotech­nology in Lithuania, pro­vided com­pu­ta­tional mod­eling exper­tise to the project. The project took advan­tage of the single-​​molecule exper­tise in the Williams lab­o­ra­tory and used a series of optical tweezers exper­i­ments to find that the DNA poly­merase sub­unit of the 10-​​subunit bac­te­rial replica­tive DNA enzyme has affinity for both double and single-​​stranded DNA in dis­tinct sub­do­mains of the protein.

DNA poly­merase III is respon­sible for copying the entire genome of E. coli every time a cell divides. The alpha sub­unit is the enzyme that actu­ally copies the DNA, and that activity is well-​​known. How­ever, there are addi­tional parts of the pro­tein that were not char­ac­ter­ized and that the researches sus­pected had DNA binding activity. The researchers first con­firmed that the pro­tein binds both double– and single-​​stranded DNA. Using pro­tein engi­neering methods to iso­late pro­tein domains, they were able to localize the two dif­ferent DNA binding activ­i­ties to two dif­ferent domains of the protein.

The single-​​stranded DNA binding com­po­nent appears to be pas­sive, because the pro­tein does not assist in melting but instead binds to single-​​stranded regions which are already sep­a­rated by force,” said Beuning. “Detecting this kind of binding would be dif­fi­cult or impos­sible using tra­di­tional methods of assaying DNA binding activity.”

The researchers’ results demon­strated that single-​​stranded DNA binding is local­ized to an OB-​​fold domain while a tandem, helix-​​hairpin-​​helix motif con­tributes sig­nif­i­cantly to double-​​stranded DNA binding. Single-​​stranded DNA binding by the sub­unit occurs only after single-​​stranded DNA has been fully melted by force. This unusual behavior, noted Beuning, may be func­tion­ally impor­tant as single-​​stranded DNA binding will likely occur only after other repli­ca­tion processes create single-​​stranded DNA.

It is cru­cial to under­stand how these kinds of mas­sive bio­log­ical machines func­tion in the cel­lular envi­ron­ment in order to fully exploit their poten­tial as drug tar­gets,” added Beuning.

For more infor­ma­tion, please con­tact Samantha Fodrowski at 617–373-5427 or at s.​fodrowski@​neu.​edu.

About North­eastern

Founded in 1898, North­eastern Uni­ver­sity is a pri­vate research uni­ver­sity located in the heart of Boston. North­eastern is a leader in inter­dis­ci­pli­nary research, urban engage­ment, and the inte­gra­tion of class­room learning with real-​​world expe­ri­ence. The university’s dis­tinc­tive coop­er­a­tive edu­ca­tion pro­gram, where stu­dents alter­nate semes­ters of full-​​time study with semes­ters of paid work in fields rel­e­vant to their pro­fes­sional inter­ests and major, is one of the largest and most inno­v­a­tive in the world. The Uni­ver­sity offers a com­pre­hen­sive range of under­grad­uate and grad­uate pro­grams leading to degrees through the doc­torate in six under­grad­uate col­leges, eight grad­uate schools, and two part-​​time divi­sions. For more infor­ma­tion, please visit www​.north​eastern​.edu.