A pathway to bypass DNA lesions in the replication process is experimentally shown

December 3, 2012
In the DNA replication process, the two strands who act as a template to synthesise a complementary strand are separated, and the new complementary strand joins each of the initial strands in order to obtain two identical copies of the original DNA molecule.

DNA lesions are really common —about one million individual molecular lesions per cell per day— because its long strands usually have one missing base or are damaged. These lesions can stall the DNA replication process, what can lead to the cell death. To avoid it, there are several pathways to bypass lesions in order to continue with the process of DNA replication. One of these processes has been entirely reproduced in vitro using some techniques of manipulation of single-molecules in a study published today in Science, led by the researcher of the University of Barcelona Maria Mañosas.

"This pathway was proposed in the seventies and now we have been able to prove it on a through the manipulation of single-molecules that, oppositely to the traditional that work with a great number of molecules, allows to study how a protein works on a molecule in real time", explains Mañosas, professor at the Department of of the UB, affiliated with the campus of International excellence, BKC.

When in any of the two derived strands there is a lesion, especially in the leading strand, the polymerase stops synthetizing the bases, so the replication process is stalled.

To study a single-molecule, we used magnetic tweezers, a technique which consists on tethering a DNA hairpin between a and a magnetic bead. A generates a magnetic field which allows manipulating the beads and generates . This system can be used in order to measure the extension changes of DNA strands through the screening of the . According to Mañosas, "proteins' activity over DNA can be inferred from the changes in the extension of the molecule. The changes are due to the proteins' work".

The template switching strategy

In the DNA replication process, the two strands who act as a template to synthesise a complementary strand are separated, and the new complementary strand joins each of the initial strands in order to obtain two identical copies of the original . In this process take part the polymerases, a family of enzymes that carry out all forms of DNA replication. When in any of the two derived strands there is a lesion, especially in the leading strand, the stops synthetizing the bases, so the replication process is stalled. "To stall this process can entail some problems in cellular growth", explains Mañosas. "When the replication mechanism (replisome) is disassembled, the bypass process analysed in this study starts", points out the author, member of the Biomedical Research Networking center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) and researcher at the University of Paris.

The action of a helicase protein (UvsW) promotes the binding of DNA strands to build an intermediate structure (Holliday junction) taking as a model the not damaged replicated strand.

The studied process begins with the action of a helicase protein (UvsW) which promotes the binding of , a phenomenon named DNA hybridization. This protein is also able to build an intermediate structure (Holliday junction) taking as a model the not damaged replicated strand and, together with the action of polymerase, drive the system to its departure point, once "jumped" the lesion, and then restart the DNA replication process. "Therefore, the information lost when one strand is damaged can be recovered from the other intact strand which acts as a backup; this process is named "the template switching strategy". In the study, we have also observed the regulation mechanisms of this pathway, as well as the rate of annealing of helicase UvsW, 1500 bases per second, one the largest known", concludes Mañosas.

The protein UvsW, together with the coordinated action of the polymerase, drives the system to its departure point, once “jumped” the lesion.

DNA repair is essential in a great number of diseases. A deeper knowledge of these phenomena will enable us to act over some proteins which have similar functions in humans. Mañosas is working on this direction; she is carrying out a study on a human protein named HARP in order to know how it works, because it is known that it has a really important role in the genome conservation and its dysfunction is related to some types of cancer.

Explore further: Nanoscopic gold spheres can be reversibly bound to DNA strands reversibly bound to DNA strands

More information: Manosas, M., et al., Direct Observation of Stalled Fork Restart via Fork Regression in the T4 Replication System. Science, 30 November 2012: 1217-1220. DOI:10.1126/science.1225437.

Related Stories

Clearing jams in copy machinery

September 19, 2005

Bacteria and humans use a number of tools to direct perhaps the most important function in cells -- the accurate copying of DNA during cell division. New research published this week in Molecular Cell from the laboratory ...

Copy number variation may stem from replication misstep

December 27, 2007

Genome rearrangements, resulting in variations in the numbers of copies of genes, occur when the cellular process that copies DNA during cell division stalls and then switches to a different genetic “template,” said researchers ...

Single-DNA images give clues to breast cancer

October 29, 2012

For the first time, researchers at the University of California, Davis, have watched single strands of DNA being prepped for repair. The research, published this week in the journal Nature, has implications for understanding ...

Recommended for you

How the finch changes its tune

August 3, 2015

Like top musicians, songbirds train from a young age to weed out errors and trim variability from their songs, ultimately becoming consistent and reliable performers. But as with human musicians, even the best are not machines. ...

4 million years at Africa's salad bar

August 3, 2015

As grasses grew more common in Africa, most major mammal groups tried grazing on them at times during the past 4 million years, but some of the animals went extinct or switched back to browsing on trees and shrubs, according ...

A look at living cells down to individual molecules

August 3, 2015

EPFL scientists have been able to produce footage of the evolution of living cells at a nanoscale resolution by combining atomic force microscopy and an a super resolution optical imaging system that follows molecules that ...

New lizard named after Sir David Attenborough

August 3, 2015

A research team led by Dr Martin Whiting from the Department of Biological Sciences recently discovered a beautifully coloured new species of flat lizard, which they have named Platysaurus attenboroughi, after Sir David Attenborough.

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.