Final Activity Report Summary - DNA REPAIR (DNA repair and human health)
DNA, the genetic material inside all cells, is being damaged continuously by simply existing in an aqueous environment inside cells. Exposure to sunlight, to environmental carcinogens, for example in the diet, and to environmental radiation substantially increases the amounts of DNA damage. In order to protect cells against the consequences of this damage, all organisms have evolved a sophisticated series of repair and response mechanisms to deal with all kinds of damage. The importance of these repair and response mechanisms can be seen by the existence of more than 10 genetic disorders, all associated with defects in the way cells repair or respond to different types of DNA damage. The elevated incidence of cancer in most of these disorders indicates that these DNA damage responses represent crucial protection mechanisms against carcinogenesis. This is, however, not the whole story. Several of these disorders have multi-system clinical abnormalities with defects in the immune system, the neurological system, and more generally in many aspects of differentiation and development. Furthermore several of the disorders are associated with features of premature ageing. Taken together this demonstrates that DNA repair plays a central role in protecting us against cancer and premature ageing, as well as ensuring proper development and maintaining us in a healthy condition.
Investigation of the DNA repair disorders not only assists afflicted individuals in diagnosis, prevention and ultimately cures, but more generally provides an improved understanding of carcinogenesis, development and ageing in the general population. In the long term this will provide new therapeutic targets and methodologies for cancer treatment, as well as improving the quality of life in old age. The three genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne Syndrome (CS), that were the focus of this proposal lie at the crossroads between DNA damage, its repair, transcription and replication. During the course of the contract we have collected new cell strains from many patients and confirmed or excluded the clinical diagnosis. We have identified two new genes that are defective in some of these patients.
The main way in which cells remove ultraviolet damage from DNA is by a process termed nucleotide excision repair (NER). The early steps in NER were worked out many years ago, but we have identified the enzymes (DNA polymerases and ligase) involved in the later steps of patching up the DNA after the damage has been cut out of the DNA. Damage in regions of DNA that are being actively transcribed is repaired preferentially by transcription-coupled repair or TCR. It has been known for a long time that patients with CS are specifically defective in TCR, and we have now discovered the reason. In TCR, the CSA and CSB proteins that are defective in CS patients are needed to recruit the main NER proteins to the site of damage. They also recruit other proteins that open up the chromosome structure so that the damage can be repaired.
A key protein in NER is TFIIH, a complicated protein which has many components, and unusually has two separate functions, in NER and in transcribing DNA into RNA. We have discovered a new tenth subunit of TFIIH and excitingly we found that it is defective in a group of TTD patients designated TTDA. We have spent several years generating a mouse in which one of the subunits of TFIIH is tagged with a yellow fluorescent protein. TFIIH is therefore fluorescently labelled in every cell in the body of this mouse. We are therefore in a position to follow the fate of TFIIH in any cell type in the body that we choose, rather than being confined to cultured cells.
Cells also need to replicate their DNA containing unrepaired damage - deficiency in this ability can result in XP. We have shown that a crucial step involves the modification of a protein clamp that recruits the appropriate enzymes that are able to replicate damaged DNA.
Investigation of the DNA repair disorders not only assists afflicted individuals in diagnosis, prevention and ultimately cures, but more generally provides an improved understanding of carcinogenesis, development and ageing in the general population. In the long term this will provide new therapeutic targets and methodologies for cancer treatment, as well as improving the quality of life in old age. The three genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne Syndrome (CS), that were the focus of this proposal lie at the crossroads between DNA damage, its repair, transcription and replication. During the course of the contract we have collected new cell strains from many patients and confirmed or excluded the clinical diagnosis. We have identified two new genes that are defective in some of these patients.
The main way in which cells remove ultraviolet damage from DNA is by a process termed nucleotide excision repair (NER). The early steps in NER were worked out many years ago, but we have identified the enzymes (DNA polymerases and ligase) involved in the later steps of patching up the DNA after the damage has been cut out of the DNA. Damage in regions of DNA that are being actively transcribed is repaired preferentially by transcription-coupled repair or TCR. It has been known for a long time that patients with CS are specifically defective in TCR, and we have now discovered the reason. In TCR, the CSA and CSB proteins that are defective in CS patients are needed to recruit the main NER proteins to the site of damage. They also recruit other proteins that open up the chromosome structure so that the damage can be repaired.
A key protein in NER is TFIIH, a complicated protein which has many components, and unusually has two separate functions, in NER and in transcribing DNA into RNA. We have discovered a new tenth subunit of TFIIH and excitingly we found that it is defective in a group of TTD patients designated TTDA. We have spent several years generating a mouse in which one of the subunits of TFIIH is tagged with a yellow fluorescent protein. TFIIH is therefore fluorescently labelled in every cell in the body of this mouse. We are therefore in a position to follow the fate of TFIIH in any cell type in the body that we choose, rather than being confined to cultured cells.
Cells also need to replicate their DNA containing unrepaired damage - deficiency in this ability can result in XP. We have shown that a crucial step involves the modification of a protein clamp that recruits the appropriate enzymes that are able to replicate damaged DNA.