DNA Repair

Damage to DNA can result from several different types of processes. Hydrolysis, deamination, alkylation, and oxidation are all capable of causing a modification in one or more bases in a DNA sequence.

Hydrolysis.

DNA consists of long strands of sugar molecules called deoxyribose that are linked together by phosphate groups. Each sugar molecule carries one of the four natural DNA bases: adenine, guanine, cytosine, or thymine (A, G, C, or T). The chemical bond between a DNA base and its respective deoxyribose, although relatively stable, is nonetheless subject to chance cleavage by a water molecule in a process known as spontaneous hydrolysis. Loss of the "purine" bases (guanine and adenine) is referred to as depurination, whereas loss of the "pyrimidine" bases (cytosine and thymine) is called depyrimidination. In mammalian cells, it is estimated that depurination occurs at the rate of about 10,000 purine bases lost per cell generation. The rate of depyrimidination is considerably slower, resulting in the loss of about 500 pyrimidine bases per cell generation.

The baseless sugars that result from these processes are commonly referred to as AP-sites (apurinic/apyrimidinic). They are potentially lethal to the cell, as they act to block the progress of DNA replication, but are efficiently repaired in a series of enzyme-catalyzed reactions collectively referred to as the base excision repair (BER) pathway. In fact, AP-sites are intentionally created during the course of BER.

Deamination.

The bases that make up DNA are also vulnerable to modification of their chemical structure. One form of modification, called spontaneous deamination, is the loss of an amino group (-NH₂). For example, cytosine (C), which is paired with guanine (G) in normal, double-stranded DNA, has an amino group attached to the fourth carbon (C4) of the base. Figure 2. If damaged DNA (*) is not repaired, the mistake (mutation) can be replicated and become permanent (fixed) in the genome. This may cause severe problems.

When that amino group is lost, either through spontaneous, chemical, or enzymatic hydrolysis, a uracil (U) base is formed, and a normal C-G DNA base pair is changed to a premutagenic U-G base pair (uracil is not a normal part of DNA).

The U-G base pair is called premutagenic because if it is not repaired before DNA replication, a mutation will result. During DNA replication, the DNA strands separate, and each strand is copied by a DNA polymerase protein complex. On one strand, the uracil (U) will pair with a new adenine (A), while on the other strand the guanine (G) will pair with a new cytosine(C). Thus, one DNA double-strand contains a normal C-G base pair, but the other double-strand has a mutant U-A base pair. This process is called mutation fixation, and the mutation of the G to an A is said to be fixed (meaning "fixed in place," not "repaired"). In other words, the cell now accepts the new mutant base pair as normal. It is estimated that approximately 400 cytosine deamination events per genome occur every day. Clearly, it is very important for the cell to repair DNA damage before DNA replication commences, in order to avoid mutation fixation. One cause of normal human aging is the gradual accumulation over time of mutations in our cellular DNA.

Alkylation.

Another type of base modification is alkylation (Figure 2C). Alkylation occurs when a reactive mutagen transfers an alkyl group (typically a small hydrocarbon side chain such as a methyl or ethyl group, denoted as-CH₃ and-C₂H₅, respectively) to a DNA base. The nitrogen atoms of the purine bases (N₃ of adenine and N₇ of guanine) and the oxygen atom of guanine (O₆) are particularly susceptible to alkylation in the form of methylation. Methylation of DNA bases can occur through the action of exogenous (environmental) and endogenous (intracellular) agents. For example, exogenous chemicals such as dimethylsulfate, used in many industrial processes and formed during the combustion of sulfur-containing fossil and N-methyl-N-nitrosoamine, a component of tobacco smoke, are powerful alkylating agents. These chemicals are known to greatly elevate mutation rates in cultured cells and cause cancer in rodents.

Inside every cell is a small molecule known as S-adenosylmethionine or "SAM." SAM, which is required for normal cellular metabolism, is an endogenous methyl donor. The function of SAM is to provide an activated methyl group for virtually every normal biological methylation reaction. SAM helps to make important molecules such as adrenaline, a hormone secreted in times of stress; creatine, which provides energy for muscle contraction; and phosphatidylcholine, an important component of cell membranes. However, SAM can also methylate inappropriate targets, such as adenine and guanine. Such endogenous DNA-alkylation damage must be continually repaired; otherwise, mutation fixation can occur.

Oxidation.

Oxidative damage to DNA bases occurs when an oxygen atom binds to a carbon atom in the DNA base (Figure 2D). High-energy radiation, like X rays and gamma radiation, causes exogenous oxidative DNA base damage by interacting with water molecules to create highly reactive oxygen species, which then attack DNA bases at susceptible carbon atoms. Oxidative base damage is also endogenously produced by reactive oxygen species released during normal respiration in mitochondria, the cell's "energy factories."

Humans enjoy a long life span; thus, it would seem that healthy, DNA repair-proficient cells could correct most of the naturally occurring endogenous DNA damage. Unfortunately, when levels of endogenous DNA damage are high, which might occur as the result of an inactivating mutation in a DNA repair gene, or when we are exposed to harmful exogenous agents like radiation or dangerous chemicals, the cell's DNA repair systems become overwhelmed. Lack of DNA repair results in a high mutation rate, which in turn may lead to cell death, cancer, and other diseases. Also, if the level of DNA repair activity declines with age, then the mutational burden of the cell will increase as we grow older.