Dna Damage And Dna Repair

> DNA in human cells is continuously subject to damage. It is in most cases appropriately repaired, leaving relatively few permanent changes. The various kinds of damage comprise chemical modification or loss of DNA bases, single strand or double strand breaks as well as intra- and interstrand crosslinks. Each type of damage can lead to mutations. An important source of mutations are DNA replication and recombination. DNA replication is a particular critical phase, during which misincorporation of nucleotides, DNA polymerase slippage or stalling of replication forks may occur. A further source of mutations are physiological recombination processes that go astray, e.g. in germ cells or lymphocytes.

> In addition to endogenous processes such as oxidative stress and spontaneous reactions of DNA such as cytosine deamination, diverse exogenous physical and chemical carcinogens cause DNA damage. Some carcinogens cause specific point mutations while others induce strand-breaks or various types of alterations. Carcinogens that induce strand-breaks may act as 'clastogens', i.e. induce structural chromosomal aberrations. The involvement of specifically acting carcinogens is in some cases detectable by the kind of mutation found in a cancer.

^ Tumor viruses can be mutagenic by insertion, by causing rearrangements or loss of chromosomes, or indirectly through viral proteins, which interfere with the cellular systems that control genomic integrity.

^ The various DNA repair systems in human cells are tailored towards the different types of DNA damage. They share components such as DNA polymerases and DNA ligases, but each employ additional specific proteins.

^ Specialized glycosylases remove damaged bases. AP (apurinic/apyrimidinic) endonucleases prepare sites lacking bases for short patch or long patch base excision repair. More problematic alterations such as carcinogen adducts and pyrimidine dimers caused by UV light are removed by nucleotide excision repair systems. Mismatched base pairs in DNA caused by mutagens or mistakes during DNA replication are the target of two interlinked mismatch repair systems. Double strand breaks pose a major challenge to cell survival and genomic integrity. They are recognized and handled by several different repair systems employing homologous or non-homologous recombination to avoid or minimize permanent damage. Still another repair system employs the FANC proteins to prepare crosslinked DNA for repair by recombination.

> Inborn errors in these DNA repair systems underlie syndromes associated with developmental defects, neurological disease and cancer. For instance, excision repair is defective in xeroderma pigmentosum, mismatch repair in HNPCC (hereditary non-poliposis carcinoma coli), double strand repair in ataxia telangiectasia, and cross-link repair in Fanconi anemia, respectively. While each of these syndromes is rare, polymorphisms in DNA repair genes likely modulate cancer risk in the general population. ^ A second layer of protective mechanisms helps to avoid DNA damage. Reactive mutagens are intercepted by low molecular protective compounds such as glutathione or by proteins such as metallothioneins or glutathione transferases. Specific mechanisms protect against reactive oxygen species and against radiation. Genetic polymorphisms again, but also diet and other environmental factors influence the efficiency of these mechanisms in individual humans. > DNA damage can activate cellular checkpoints which prevent cell cycle progression and stop DNA synthesis and mitosis, or activate apoptotic cell death. Double-strand breaks elicit a particularly strong signal. Stress signals can also be activated by radiation or reactive oxygen species, through specialized signaling pathways. Infection by viruses also activates cellular checkpoints and stress signals.


The mutations and chromosomal alterations found in cancer cells (^2) represent only a small fraction of those that arise during the life-time of a human, because the great majority are removed by one of several repair mechanisms (Table 3.1). These are excellently tuned to the various types of DNA damage that might cause mutations (Figure 3.1). Moreover, cells with substantially damaged DNA or aneuploid genomes are normally eliminated or at least prevented from proliferation. Therefore, cancer cells displaying genomic instability need to inactivate the systems responsible for this surveillance. Accordingly, defects in DNA repair systems and cellular surveillance mechanisms are an important, if not even necessary factor in the development of human cancers. Such defects may be inherited or acquired.

Damage to DNA can result from endogenous as well as exogenous sources. DNA replication is a particularly critical process, with an increased potential for spontaneous mutations and an increased sensitivity towards induced damage. Proliferating cells are therefore more susceptible to neoplastic transformation. Problems that may arise during DNA replication comprise misincorporation of bases, slippage of the replisome in tandem repeat sequences, single-strand breaks being converted into double-strand breaks by replication, stalling of the replisome at 'difficult' sequences or at bases modified by chemical reactions with exogenous carcinogens or endogenous proteins.

Replication of nuclear DNA is extremely precise with a nucleotide misincorporation rate of 10-7- 10-6, since eukaryotic replication DNA polymerases discrimate well between the various nucleotides and the main replicase possesses a 3'-5' exonuclease proof-reading function. This level of precision is not always achieved by repair polymerases. In spite of the excellent fidelity of the replication proteins, in a genome of >3 x 109 bp, several hundred mistakes are expected during each replication. Most misincorporations are corrected by base mismatch repair systems, leaving an estimated number of 1 x 10-10 base changes per cell division.

Base misincorporation is, however, only one of several problems that can occur during replication. Mismatch repair (MMR) systems also take care of single strand loops in replicated DNA that result from slippage of DNA in repeat sequences. Typically, slippage occurs in microsatellites which consist of tandem repeats of 1-4 bp repeats. Defects in mismatch repair therefore result in an increased frequency of base misincorporations, but also lead to microsatellite expansions or contractions. Even with fully functional mismatch repair, microsatellites are subject to a somewhat higher mutation rate than the average of the genome, which is one reason why they are normally polymorphic.

Another source of base misincorporation are mesomeric isoforms of the DNA bases that can mispair. The mesomeric isoforms of the standard four bases are shortlived, but some are stabilized by chemical modification. For instance, hydroxylation of guanine at the 8 position stabilizes a G:A mismatch. Most mismatches caused from frequent mispairing events such as OH-G:A are recognized by specific proteins that activate the mismatch repair system.

The precision of DNA replication also depends on the nucleotide precursor pools. Disparities in the relative levels of the deoxy-nucleotide triphosphates decrease the fidelity of base incorporation. As biochemistry textbooks discuss in detail, nucleotide biosynthetic pathways contain several crossregulatory and

Table 3.1. An overview of DNA repair mechanisms in human cells

Type of DNA damage Repair Mechanisms

Selected proteins involved

Base misincorporation Mismatch Repair

Chemical modification of bases Base loss

Base excision + short patch repair Base excision + short patch repair Short patch repair

Formation of intrastrand Nucleotide excision repair base dimers or bulky adducts at bases

Bypass repair Interstrand crosslinks Crosslink repair

Double-strand breaks Homologous recombination repair

Nonhomologous DNA end joining


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