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Retrospective analysis of double-strand break rejoining data collected using warm-lysis PFGE protocols.

Abstract：

:Sample preparation procedures for the pulsed-field gel electrophoresis (PFGE) assay usually involve a lysis step at temperatures as high as 50 degrees C. During this warm-lysis procedure, multiply damaged sites containing heat-labile sites (HLS) can be converted into double-strand breaks (DSB). Once formed, these DSB cannot be distinguished from the DSB formed directly by ionizing radiation. This paper develops a method to correct DSB estimates for the effects of HLS in warm-lysis protocols. A first-order repair model is used to predict the number of HLS available for conversion into DSB as a function of the time available for repair before initiating warm-lysis. A mathematical expression is derived to separate prompt DSB from those formed through the artefactual conversion of HLS into DSB. The proposed formalism only requires the specification of two adjustable parameters, both of which can be estimated from measured data. Estimates of prompt DSB yields obtained by correcting warm-lysis data are in good agreement with estimates obtained using cold-lysis protocols, which do not include the effect of HLS. The retrospective analyses of two published datasets suggest that corrections for HLS have a substantial impact on DSB yields within the first 20-30 min after irradiation. Bi-exponential fits to the DSB data for Chinese hamster ovary cells suggest that corrections for HLS reduce the half-time for fast DSB rejoining by about 15%, whereas the half-time for the slow DSB rejoining only decreases by 4%. The total DSB yield and the fraction of fast-rejoining DSB decrease by 24 and 38%, respectively, when the correction is applied. The proposed formalism can be used to characterize trends and uncertainties in DSB rejoining kinetics associated with the artefactual conversion of HLS into DSB. The retrospective application of the methodology to warm-lysis data enhances their relevance and usefulness for studies of DSB rejoining kinetics.

journal_name

Int J Radiat Biol

journal_title

International journal of radiation biology

authors

Ratnayake RK,Semenenko VA,Stewart RD

doi

10.1080/09553000500156577

subject

Has Abstract

pub_date

2005-06-01 00:00:00

pages

421-8

issue

eissn

0955-3002

issn

1362-3095

pii

Q7G0VJ6695437268

journal_volume

pub_type

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Retrospective analysis of double-strand break rejoining data collected using warm-lysis PFGE protocols.