Cells Can Read Damaged DNA Without Missing a Beat
ScienceDaily (Feb. 10, 2010) — Scientists have shown that cells' DNA-reading machinery can skim through certain kinds of damaged DNA without skipping any letters in the genetic "text." The studies, performed in bacteria, suggest a new mechanism that can allow bacteria to develop resistance to antibiotics.
Working with Doetsch, graduate student Cheryl Clauson examined the ability of RNA polymerase (the enzyme that transcribes, or makes RNA from DNA) to handle damaged DNA templates.
RNA polymerase reads one strand of the double helix and assembles RNA that is complementary to that strand. In test tube experiments, when the enzyme comes to a gap or a blank space, it keeps reading but leaves out letters across from the damaged stretch. In contrast, in cells, RNA polymerase puts a random letter (preferring A) across from the gap.
"We were surprised to find that the transcription machinery rolls right over the damaged portion," Doetsch says. "This shows that if the cell initiates, but doesn't complete repair, it still can lead to mutagenesis."
Clauson says a challenge in planning her experiments was finding a way to sensitively detect when RNA polymerase reads through DNA damage.
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Abasic sites and strand breaks in DNA cause transcriptional mutagenesis inEscherichia coli
Cheryl L. Clauson a,b, Kenneth J. Oestreich c,1, James W. Austin b,c, and Paul W. Doetsch a,d,e,f,2
-Author Affiliations
Departments of a Biochemistry,
cMicrobiology and Immunology,
dRadiation Oncology, and
eHematology and Medical Oncology,
fWinship Cancer Institute, and
bGraduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, GA 30322
↵1Present address: Department of Immunology, University of Washington, Seattle, WA 98195.
Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved January 15, 2010 (received for review November 13, 2009)
Abstract
DNA damage occurs continuously, and faithful replication and transcription are essential for maintaining cell viability. Cells in nature are not dividing and replicating DNA often; therefore it is important to consider the outcome of RNA polymerase (RNAP) encounters with DNA damage. Base damage in the DNA can affect transcriptional fidelity, leading to production of mutant mRNA and protein in a process termed transcriptional mutagenesis (TM). Abasic (AP) sites and strand breaks are frequently occurring, spontaneous damages that are also base excision repair (BER) intermediates. In vitro studies have demonstrated that these lesions can be bypassed by RNAP; however this has never been assessed in vivo. This study demonstrates that RNAP is capable of bypassing AP sites and strand breaks in Escherichia coli and results in TM through adenine incorporation in nascent mRNA. Elimination of the enzymes that process these lesions further increases TM; however, such mutants can still complete repair by other downstream pathways. These results show that AP sites and strand breaks can result in mutagenic RNAP bypass and have important implications for the biologic endpoints of DNA damage.
BER DNA repair AP lyase AP endonuclease RNA polymerase
Footnotes
2To whom correspondence should be addressed. E-mail:medpwd@emory.edu.
Author contributions: C.L.C. and P.W.D. designed research; C.L.C., K.J.O., and J.W.A. performed research; C.L.C. and P.W.D. analyzed data; and C.L.C. and P.W.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0913191107/DCSupplemental.
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