Repair replication

It is important to have an experimental procedure that provides direct evidence that the new incorporation seen after insult is really in parental DNA (i.e., represents repair of previously existing DNA). 

In repair replication experiments, cells are irradiated and then incubated in medium containing 3H-5-bromodeoxyuridine ( H-BrdUrd). After a suitable repair period the cells are harvested, and the DNA is extracted and centrifuged to equilibrium in cesium chloride. When the gradient fractions are collected, it is possible to follow the new DNA (semiconservatively synthesized during repair and at hybrid density) by counts. The new DNA is half substituted with bromouracil (BrUra) and sediments to a lower point on the gradient than the old or parental DNA, which is detected by absorbancy at 260 nm or by incorporation of a second radioactive label and is, of course, not density labeled. The appearance of counts in the old DNA region is indicative that repair has occurred and that the new incorporation is truly in the parental 

Furthermore, if the initial number of DNA lesions or breaks induced by the insult (as in the case of ionizing radiation) and the specific activity of the H-BrdUrd are known, it is possible to calculate the average number of base insertions per repaired region (Painter and Young 1972). A diagrammatic representation of the result of a typical repair replication experiment is shown in Fig. 1. 

Billen s (1969) studies of DNA repair in Escherichia coli after X-irradiation suggest that most of the repair synthesis seen after irradiation is not base insertion into regions being repaired but represents new growing points in the DNA. On the basis of thymidine uptake experiments and the other repair assays discussed above, it is quite unlikely that this is the case for human cells. In Go peripheral leukocytes, for example, there is little semiconservative synthesis, and the hydroxyurea present would presumably inhibit any induction of new growing points. 

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Robison SH. Cantoni 0, Costa M. 1984. Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells. MutRes 131 173-181. 

Meneghini R. 1974. Repair replication of opossum lymphocyte DNA Effect of compounds that bind to DNA. Chem Biol Interact 8 113-126. 

Craddock VM, Flenderson AR. 1978. De novo and repair replication of DNA in liver of carcinogen-treated animals. Cancer Res 38 2135-2143. 

Pettijohn, D.E. Hanawalt, P.C. (1964). Evidence for repair-replication of ultra-violet damaged DNA in bacteria. J. molec. Biol. 9, 395-410. 

Biological activity Replication DNA repair Replication Replication Replication... 

Cleaver, J.E. (1968) Defective repair replication of DNA in xeroderma pigmentosum. Nature, 218, 652-656. 

This book is divided into two parts. The focus of Part One is on the chemical aspects of DNA damage, while the emphasis of Part Two is on the structural and functional relationships of DNA lesions, and their processing by the cellular machineries of repair, replication, and transcription. Chapter 1 in Part One is intended as a brief overview of the vast field of DNA damage, and introduces the reader to the relationships between the chemical and structural aspects of DNA damage, and some of the known biological endpoints and correlations with human disease. Ample references are provided with an emphasis on authoritative, recently published reviews to guide the interested reader. 

Another postreplication repair system is that of mismatch repair, which can repair replication mistakes that escape proofreading or which arise from chemical alteration of bases, such as deamination of cytosine to form uracil. 

PARP-1 Interacts with DNA Repair/Replication Proteins... 

A human hereditary disease, xeroderma pigmentosum, is inherited as an autosomal recessive. It is characterized by the patient s extreme sensitivity to ultraviolet light (UV) and subsequent development of skin abnormalities, some of which may become malignant. Tissue culture cells derived from a homozygous recessive individual were more sensitive to UV than those from normal subjects (Gartler, 1964). Cultured cells from other xeroderma patients exhibit minimal DNA repair replication after UV (Cleaver, 1968), because the cells cannot perform the first step (excision... 

DNA single-strand break frequency was measured by alkaline elution [16] and sister chromatid exchanges (SCEs) were assayed by the method described by Wolff [32]. Hypoxanthine-guanine phosphoribosyl transferase (HPRT 6-thioguanine) and Na/K ATPase (ouabain) resistant mutants of CHO were determined by the methods described by Cleaver [7]. Repair replication after MMS treatment was measured in isopycnic gradients [8]. 

Isopycnic gradients of bromodeoxyuridine plus [ H]-thymidine-labeled DNA from cells exposed to MMS separated the DNA into heavy semiconservatively replicated DNA and normal density repair replicated DNA. The amount of repair in WIL-2 cells exposed to MMS was strongly dependent on both MMS dose and SAB concentration (Fig. 3a,b). At low concentrations of SAB there was a sharp increase in repair replication which saturated at 2 mM (Fig. 3b). 

Fig. 3. a Repair replication in WIL-2 cells grown in C]-thymidine, then exposed to increasing concentrations of MMS and grown for 4 h with ( ) or without (9) 5 mM SAB. b Repair replication in WIL-2 cells exposed to 4 mM MMS and grown for 4 h with various concentrations of SAB. (From Cleaver [8])... 

Repair replication after MMS exposures in WIL-2 lymphoid cells was increased sevenfold by addition of SAB (Fig. 4, [8]). This could be caused by an increased number of repaired patches or increased patch sizes. An increased number of patches at sites of original damage is unlikely because SAB did not influence the excision of alkylated bases [9, 12]. Determination of the length of the repair patch indicated that SAB had no effect on patch size [8]. This observation is inconsistent with the notion that SAB causes longer patches to be synthesized because of inhibition of ligase II. Instead, there must be a greater number of patches which are not at sites of initial damage. 

Cleaver JE (1985) Increased repair replication in human lymphoid cells by inhibition of polyadenosine diphosphoribose synthesis with no increase in patch sizes. Cancer Res 45 1163-1169... 

Cd(II) (4pM) blocked UV-induced unscheduled DNA synthesis (i.e., repair replication) in human cells, and this inhibition was blocked by a higher molarity of Zn(II) (Nocentini 1987). The same concentration of Cd(II) caused the accumulation of DNA strand breaks, and this was also blocked by a tenfold molar excess of Zn(II) (Nocentini 1987). Because the initial decrease in size of DNA from UV-irradiated cells was not affected by Cd(II), it is unlikely that the initial incision steps of excision repair were inhibited. However, a more toxic treatment of cells with CdCl2 did inhibit pyrimidine dimer removal (Snyder et al. 1989). UV-induced cytotoxicity was increased by Cd(II), an effect also blocked by Zn(II). Taken together, these results suggest that Cd(II) and Zn(II) compete for a common binding site on enzymes involved in DNA replication and repair. Cd(II) inhibits repair of X-ray-induced strand breaks (DNA ligase ) (Snyder et al. 1989). 

Robison SH, Cantoni O, Costa M (1984) Analysis of metal-induced DNA lesions and DNA repair replication in mammalian cells. Mutat Res 131 173-181 Rodney PF, Robert JJ, Lay PA, Dixon NE, Raker RSU, Bonin AM (1989) Chromium (V)-induced cleavage of DNA are chromium (V) complexes the active carcinogens in chromium (Vl)-induced cancer Chem Res Toxicol 2 227-229... 

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