TITLE: ROLE OF NUCLEOTIDE EXCISION, DEFICIENCY AND
OTHER MECHANISMS OF DNA REPAIR
The huge variety of DNA –
reactive chemicals in our environment combined with the huge variety of
alterations that can be produced by radiation and by oxidative and free radical
attack on DNA can generate so many types of damage that coping with all types
of damage by evolutionary development of damage-specific DNA glycosylase would
be difficult if not impossible. Fortunately, a different, more flexible damage
repair mechanism has evolved in living organisms, NUCLEOTIDE EXCISION REPAIR
(NER) which recognizes damaged regions based on their abnormal structure as
well as on their abnormal chemistry, then excises and replaces them.
DNA damage recognition. Two proteins have been
identified and implicated in (one of) the first steps of NER, i.e. the
recognition of lesions in the DNA: the XPA gene product and the XPC gene
product in complex with HR23B. In addition, the XPE protein has been shown to
have a high affinity for damaged DNA, but whether it is required for the damage
recognition step of NER remains unclear. Cells from XPA patients are extremely
sensitive to UV and have very low nucleotide excision repair activity. In vitro
the XPA protein binds preferentially to damaged DNA compares to nondamaged DNA.
The XPA protein binds to replication protein A (RPA), which enhances the
affinity of XPA for damaged DNA and is essential for NER. The other complex
that has been implicated in DNA damage recognition is XPC-HR23B. XPC cells have
low NER repair capacity, but the residual repair has been shown to occur
specifically in transcribed genes. It is very likely that the XPC-HR23B complex
is the principal damage recognition complex i.e. essential for the recognition
of DNA lesions in the genome. Binding of XPC-HR23B to a DNA lesion causes local
unwinding, so that the XPA protein can bind and the whole repair machinery can
be loaded onto the damaged site. This would imply that the XPA protein has binding
affinity for the repair proteins. Indeed, the XPA protein has been shown to
bind to ERCC1 and TFIIH. The XPC-HR23B complex is only require for global
genome repair. In case of transcription coupled repair when an RNA polymerase
is stalled at a lesion, the DNA is unwound by the transcription complex and XPA
can bind independently of XPC-HR23B complex.
XPE patients show mild dermatological symptoms and
cells from these patients have a relatively high repair capacity. The function
of the gene product is not completely clarified yet. Band shift assays
suggested that the XPE gene product acts as a damaged DNA binding protein
(DDB), with high affinity to UV-induced 6-4PP. However, defective DDB binding
activity is not a common feature of XPE mutant cell lines and in fact two (or
even more) proteins may be involved in the binding activity: p48 and p125. In
cells from several XPE patient mutations in have been found but so far no
mutations have been found in the p125 gene. XPE cells are not necessarily
defective in repair: p125 is proposed to play a role in opening up chromatin to
make CPD accessible to the NER machinery, but is not required for repair of
6-4PP. Interestingly, cell lines and primary tissues from rodents are fully
deficient in the expression of the p48 protein. This explains the absence of
GGR of CPD in these cells. Exogenous expression of p48 in hamster cells confers
enhanced removal of CPD from genomic DNA and nontranscribed strand of active
genes.
Damage demarcation. The striking discovery that
subunits of basal transcription factor TFIIH were involved in NER sheds light
on a new aspect of NER: a close coupling to transcription via common use of
essential factors. Two repair proteins, encoded by XPB and XPD genes, appeared
to be identical to components of the basal transcription factor TFIIH, a large
complex involved in the initiation of trancription. The XPB and XPB proteins
displayed 3’-5’ and 5’-3’ helicase activity respectively. TFIIH fulfills a dual
role in transcription initiation and NER and the role of TFIIH in NER might
closely mimic its role in the transcription initiation process. In
transcription initiation TFIIH is thought to be involved in unwinding of the
promoter site and to allow promoter clearance. In the NER process TFIIH causes
unwinding of the damage containing region that has been localized by XPC-HR23B
and XPA-RPA, enabling the accumulation of NER proteins around the damaged site.
Among the XP patients, XPB patients are extremely
rare (only 3 patients known in the world) due to the fact that the XPB gene
product is essential for transcription initiation and in all cases, these
patients show the double symptoms of XP and CS. The helicase activity of XPD is
indispensable for NER but not for transcription initiation. So, there is much
more XPD patients, and only two patients have been described as XP and CS.
Incision. The XPF protein and the ERCC1 protein form a complex
that exhibits structure specific endonuclease activity that is responsible for
the 5’ incision during the NER reaction. XPF-ERCC1 also binds to XPA (through
ERCC1) and to RPA (through XPF) but not preferentially to damaged DNA. The XPG
protein has DNA endonuclease activity without preference for damaged DNA and is
responsible for the 3’ incision made during NER. At the site of a lesion NER
proteins create a DNA bubble structure over a length of approximately 25
nucleotides and the XPG protein incises the damaged DNA strand 0-2 nucleotides
3’ to the ssDNA-dsDNA junction. In most studies the 3’ incision made by the XPG
protein appeared to be made prior to and independently of the 5’-incision by
XPF-ERCC1. Patients belonging to the XPG complementation group clinically
exhibit heterogenous symptoms., from mild to very severe, sometimes associated
with CS. XP-G cells are almost completely repair-deficient and as UV- sensitive
as XP-A cells. About half of the described XPG patients exhibit also CS
symptoms. In contrast to XPG, XP-F patients have a relatively mild XP phenotype
without neurological abnormalities. Cells from XP-F patients are slightly
UV-sensitive and exhibit low levels of repair initially after UV-irradiation.
Repair patch synthesis and ligation. Proliferating Cell Nulear
Antigen (PCNA) is required for DNA synthesis by DNA polymerases delta and
epsilon. PCNA has also been shown to be required for NER in vitro i.e. for the
DNA resynthesis step, suggesting that DNA polymerase delta or epsilon is
involved in NER. Biochemical analysis and fluorescence microscopy revealed that
in quiescent cells upon UV-irradiation PCNA (that usually resides in the
cytoplasm) becomes rapidly bound to chromatin. The enzymes involved in these
pathways are normal in DNA repair-deficient cells.