COSTOS DE INGENIERÍA

Preferential binding to alkyl-dam aged D N A

XPC-hHR23B w as found to have at least a 6-fold higher preference for binding to alkyl dam aged DNA than non dam aged DNA and a 3-4 fold preference was found for UV-DDB. Binding of XPC-hHR23B to this type of DNA damage has not been studied previously, b u t NER can act w eakly on 0 6-m ethylguanine lesions - which are produced by MNU and to a lesser extent MMS and so some affinity for this type of dam age is to be expected. Payne and C hu show ed that UV-DDB b inds N 6-m ethyladenine and C5-m ethylcytosine residues w ith an affinity 400 and 1000-fold less than for (6-4) photoproducts respectively (Payne and Chu, 1994). Interaction of UV-DDB w ith 0 6-m ethylguanine or other lesions induced by MMS or MNU treatm ent h ad n 't been tested. In m y assay, UV-DDB h ad a 3-4-fold higher affinity for M M S/M N U d am ag ed DN A than non dam aged which is in the order of 100 to 150-fold less than its apparent affinity for UV dam aged DNA. It appears XPC-hHR23B and UV-DDB have similar levels of affinity for alkyl dam aged DNA. It m ust be stressed that the results presented on preferential binding of XPC-hHR23B and UV-DDB to both alkyl and platin u m dam aged DNA are as yet only prelim inary. Further w ork is req u ired u n d e r conditions w here lesion yield in the com petitor DNA is accurately m easured and also using com petitor DNA w ith single defined lesions to give more direct evidence for these interactions.

L o w affin ity f o r p o ly (dl^dC) syn th etic copolym er

The affinity of XPC-hHR23B for poly (dI»dC) is basically the same as for non dam aged double-stranded plasm id DNA. Inclusion of betw een 10-100 ng poly (dI»dC) in reaction mixtures was necessary to allow specific dam age binding to occur. W ithout poly (dI«dC) present, all of the pro b e-p ro tein complexes aggregated in the wells of the gel. UV-DDB ap p ears less sensitive to the absence of poly (dI«dC), probably due to its higher affinity for UV dam aged

DNA. Binding of UV-DDB to poly (dI«dC) is in the same range or m ay actually be slightly higher than its binding to non dam aged plasm id DNA.

High affin ity f o r heat-denatured and single-stranded M13 D N A

XPC-hHR23B appears to bind strongly to heat d en atu red (single-stranded) DNA. Experim ents using M13 DNA allow ed quantitation of the affinity of XPC-hHR23B and UV-DDB for both non and UV dam aged single-stranded M13 DNA. It appears that both proteins recognize and bind to ss M13 and bind it w ith higher affinity w hen it contains UV dam age. XPC-hHR23B shows a 70- fold higher affinity for non dam aged M13 than non dam aged ds DNA, whereas UV-DDB shows only a 25-fold higher affinity for ss M13 over ds non dam aged DNA. UV dam age in M13 ss DNA is at least as well recognized by XPC- hHR23B as it is in ds DNA. However, UV-DDB has an affinity for UV damage in ss M13 DNA 3-fold less than in ds DNA. The results presented here are in

qualitative agreem ent w ith the work of R eardon et al w ho show ed binding of

XPC-hHR23B to a single-stranded oligonucleotide in a m obility-shift assay (Reardon et al., 1996). Previous w ork by Payne and C hu show ed UV-DDB bound to ss DNA and slightly better to UV dam aged ss DNA (Payne and Chu, 1994). The w ork presented here is in agreem ent w ith this data b u t adds further quantitation of the affinity of UV-DDB (and XPC-hHR23B) for dam age in single and double-stranded DNA.

The NER process acts on a w ide variety of lesions and so it is likely that the

dam age recognition com ponents recognize a comm on feature of these. The

presence of a lesion in duplex DNA is know n to cause some local distortion and possibly the generation of a partially opened single-stranded region. Generally, the m ore helix-distorting a lesion is, the m ore efficiently it is repaired by NER and so a current hypothesis is that it is this distorted duplex structure that is the m ain d eterm in an t for recognition. The fact th at b o th XPC-hHR23B and UV-DDB appear to bind UV dam age in both single and double-stranded DNA

Chapter 6. DNA binding activities ot Xlje-hHl<23b and UV-DDB.

introduces a puzzle as to w hat exactly is being recognized. It is possible that these factors bind directly to lesions b u t as they both have affinity for several different types of DNA dam age this seem s unlikely. XPC-hHR23B and UV-DDB bind strongly to UV dam aged single-stranded M13 DNA and so a distorted DNA duplex appears not to be the m ain determ inant for recognition. However, M l3 DNA is probably not completely single-stranded as it is DNA of m ixed sequence and so is very likely to contain som e level of secondary structure. It m ay be that single-stranded M13 can actually form structures in these regions of secondary structure similar to those form ed in dam aged duplex

DNA. It is also possible th at these factors in te rac t w ith the p artial

single-strandedness present at a dam aged site in duplex DNA and therefore bind avidly to both non and UV dam aged single-stranded DNA.

The single-stranded binding activity of XPC-hHR23B and to a lesser extent UV-DDB m ay give us an im portant clue to how a dam aged site is recognized. The h ig h er level of b in d in g to non an d UV d am ag ed ss M13 DNA by XPC-hHR23B is also the m ain difference observed in the DNA binding activities of these two factors. For these reasons further w ork was carried out to attem pt to characterize XPC-hHR23B/s affinity for different types of single-stranded DNA and this is presented and discussed in chapter 7.

Chapter 7. Single-stranded DNA binding activity