Replication of DNA damage, and the role of the SMC5-6 protein complex in the responses to DNA damage

The genetic information, which determines the precise functions of every cell in the body, is contained in the sequence of the three billion nucleotide bases that make up the DNA molecules in the 46 chromosomes of every human cell. Our DNA is being continually damaged by exposure to carcinogenic chemicals and sunlight. Fortunately, we have evolved a whole series of DNA repair and other damage response mechanisms, to enable us to withstand almost every kind of DNA damage and thereby maintain the integrity of the genome.

We know that DNA repair is important for human health because there are several genetic diseases that are caused by defects in one or other of these DNA repair processes. Individuals with xeroderma pigmentosum (XP) are unable to deal with damage inflicted in DNA by ultraviolet light from the sun. As a consequence, they develop skin cancers at a frequency some 1000-fold above that in the general population.

Before a cell divides, it must replicate its DNA precisely, i.e. all of the three billion bases must be correctly copied. When DNA is damaged, most of the damage is repaired, but repair can be quite slow, and a rapidly growing cell is faced with the problem of having to replicate damaged DNA. Enzymes that replicate DNA are called DNA polymerases, and there is a recently discovered class of these DNA polymerases, which have special properties that enable them to replicate DNA containing damage. Whereas the majority of XP patients simply cannot remove damage from their DNA, a substantial minority, known as XP variants, is deficient in one of these special polymerases. The aim of this proposal is to understand how these special polymerases work inside the cell, what happens in XP variant cells when one of the polymerases is absent, and what the broader implications are for carcinogenesis in general. These special polymerases might prove to be suitable targets for chemotherapeutic drugs, and understanding how they work will assist in rational drug design.

The SMC proteins are a family of proteins that maintain the structure of the chromosomes, during the many complicated transactions that occur in the cell. One of these family members is also involved in repairing DNA damage and we are trying to understand how these proteins work.

Replication past unrepaired damage utilises a complex series of mechanisms in eukaryotic cells. Translesion synthesis (TLS) is a major pathway for replicating past DNA damage and is effected by the Y-family of DNA polymerases (pols). Pol eta is required to replicate past UV damage and its absence in xeroderma pigmentosum variants results in very high frequencies of sunlight-induced skin cancers. The polymerase sliding clamp PCNA is mono-ubiquitinated when the replication fork stalls at DNA damaged sites and this increases its affinity for Y-family pol eta, providing a model for polymerase switching. We will (1) dissect the regulation of PCNA ubiquitination and de-ubiquitination; (2) generate forms of PCNA that cannot be ubiquitinated or that are permanently ubiquitinated and examine the effects of these alterations on the responses to DNA damage; (3) microdissect the events at the stalled replication forks, using in vitro systems and fluorescent lifetime imaging; (4) analyse the biological significance of our recent collaborative discovery of ubiquitinated forms of pol eta and iota; (3) determine which proteins are involved in a back-up process that deals with UV damage in the absence of pol eta. We have shown that Y-family pol kappa is unexpectedly involved in nucleotide excision repair. We will identify the precise role of pol kappa in NER and examine its ubiquitination and stability.

The Smc5-6 complex is involved in several responses to DNA damage. Four non-SMC proteins are associated with Smc5 and 6 in the complex from S. pombe. We will (1) examine the ubiquitination activity of the complex and its interaction with DNA; (2) identify components of the mammalian complex and other interacting proteins; (3) analyse the biological functions of the complex using mutant mice and siRNA methodology.