Joining Hands to Solve a DNA Replication Puzzle

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Joining Hands to Solve a DNA Replication Puzzle

16 Apr 2005 Medical News Today

Two heads and three tools are better than one. A

Medical Institute (HHMI) professor and a colleague who mentors HHMI-

supported undergraduates in his structural biology lab are using the

tools of molecular biology, biochemistry, and biophysics to solve a

scientific puzzle.

What has their attention is the mysterious mechanism that enables DNA

replication in simian virus 40 (SV40), a mammalian model for that

vital process. " We've taken what began as a biochemical and molecular

genetic approach, then used structural biology to learn about protein

interactions, and then returned to biochemistry to validate our

structural model in a functional way, " said Ellen Fanning, an HHMI

professor at Vanderbilt University in Nashville, Tennessee. HHMI

professors are accomplished research scientists who are working to

bring the excitement of research to undergraduate teaching.

Fanning and Walter Chazin, director of Vanderbilt's Center for

Structural Biology, report their findings in the April 2005 Nature

Structural & Molecular Biology, published online March 27, 2005.

" Walter and I started working together four years ago, " Fanning

explained. " He's from a very biophysical culture, while I take a

molecular biology, biochemistry approach. It required an investment

of effort to learn each other's languages and persuade our labs to

communicate. " She said the payoff from those investments has

begun. " This paper is the first step in what will become a series of

discoveries. "

Fanning's HHMI professorship and Chazin's participation in her HHMI-

supported undergraduate research program play important roles in

their collaboration. " We have about a half dozen undergraduates and a

couple of graduate students and postdocs who are actively involved in

projects between the two labs, " Fanning said. " A lot of the mutations

and constructs are being made by undergraduate students. " She

predicted that each undergraduate will emerge from this collaboration

as an author on a published research paper.

In the research reported in Nature Structural & Molecular Biology,

the scientists sought the mechanism by which single-stranded DNA

(ssDNA) breaks free from the chains of its binding protein to allow

repair or replication, a process that is not well understood. Fanning

and Chazin found structural and biochemical evidence for that

mechanism, providing a model of this early step in DNA processing in

mammalian cells.

Every organism has an ssDNA-binding protein for DNA replication and

repair pathways. In eukaryotes or organisms whose cells have a

nucleus, it is called replication protein A (RPA). One of the common

functions of RPA in DNA processing pathways is facilitating " hand-

off, " a process that ensures that the correct proteins move into

place along the ssDNA to begin DNA processing.

RPA plays an important protective role for ssDNA. " You don't want to

have naked single-stranded DNA lying around in a cell, " explained

Fanning. " It will get tangled, make hairpins within itself, get

chewed up by nucleases. Ss binding proteins keep ssDNA straight and

accessible to the right processing enzyme. "

RPA binds with at least a dozen different repair and replication

proteins. The question has been how RPA gets dislodged, allowing

various enzymes access to the DNA for necessary processing. Fanning

and Chazin have developed a working model to answer that question.

Using SV40 as a model system, the scientists mapped atomic level

interaction on the surfaces of proteins involved in DNA processing.

They used biochemical and genetic tools to determine how the

interactions of those proteins promote synthesis of small segments of

RNA known as primers, which are required for initiation of DNA

replication.

In the SV40 system, three key proteins interact. The viral protein T

antigen (Tag) interacts with RPA and an enzyme known as DNA

polymerase-primase (pol-prim). Tag is a helicase, or DNA unwinding

enzyme. After it has unwound the DNA, it also places the pol-prim on

the DNA to make primers. The researchers studied this last step: how

Tag pulls RPA away sufficiently to load the pol-prim onto the DNA,

allowing it to synthesize primers.

Fanning and Chazin showed that interaction between Tag and RPA

requires multiple contact points. They found that, along with a

domain on RPA called RPA70, a second one, RPA32C, also needs to bind

to Tag before processing can begin.

The scientists suggest that Tag associates first with RPA32C and then

with RPA70 as the RPA molecule sits on ssDNA. Binding at both of

these points alters the conformation of RPA, scrunching it up to

expose a small stretch of ssDNA. Tag brings with it pol-prim, which

is deposited in the short stretch of unbound ssDNA. Once pol-prim is

in place, Tag and RPA are no longer needed, so they are displaced as

the third protein begins its work on the ssDNA. This is the " hand-

off. "

" This provides a testable model for how the ssDNA binding protein can

be displaced from single-stranded DNA to allow a DNA processing

pathway, " Fanning said. " This is a general phenomenon that happens

throughout all DNA processing pathways. "