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Did You Know? DNA crosslinks also interfere with another vital cellular process: transcription of genes by RNA polymerase. |
When it was discovered that DNA is the macromolecular carrier of essentially all genetic information, it was assumed that DNA is extremely stable. Consequently, it came as something of a surprise to learn that DNA is actually unstable and subject to continual damage. When DNA damage is severe, the cell is unable to replicate and may die. Repair of DNA must be regarded as essential for the preservation and transmission of genetic information in all life forms. In this article, we will discuss various types of DNA damage and the DNA repair systems that have evolved to correct that damage.
Sources of Damage
DNA is subject to spontaneous instability and decay. In addition to spontaneous damage, cellular DNA is under constant attack from reactive chemicals that the cell itself generates as by-products of metabolism. Moreover, the integrity of cellular DNA is assaulted by such environmental threats as X rays, ultraviolet radiation from the sun, and many chemical agents, some of which are products of our industrialized society.
Since mutations can be introduced into DNA as a consequence of DNA damage, there is currently great interest and concern about the expanding list of chemicals released into the environment. In humans, damage to DNA has been implicated in many cancers as well as in certain aspects of aging. Genetic diseases such as cystic fibrosis and sickle cell disease can be caused by a single DNA mutation in one gene.
Types of Dna Damage
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.
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.
Base Excision Repair
DNA bases that have been modified by the addition or loss of a small chemical group as described above are repaired by the BER pathway (Figure 3). The BER pathway begins with the excision of a damaged base by an enzyme called DNA glycosylase (Figure 3, step 1). DNA glycosylases bind to chemically altered (damaged) bases and catalyze the cleavage (hydrolysis) of the bond linking the modified base to its sugar, which results in the release of the modified base from the DNA chain and in the insertion of an AP-site. Several types of DNA glycosylases exist, each one specifically excising a different type of damaged base. It is important that a DNA glycosylase act only on damaged and not natural DNA bases, otherwise too many baseless sugars would be produced, weakening the integrity of the DNA chain.
Excision of the damaged base by a DNA glycosylase creates an AP-site, which in turn is acted upon by the second enzyme in the BER pathway, apurinic/apyrimidinic (AP) endonuclease (Figure 3, step 2). The most abundant AP-endonuclease in human cells cleaves (incises) the sugar-phosphate backbone on the left side of the baseless sugar to yield a one-nucleotide gap. On the left margin of the incision is a normal nucleotide (DNA base + sugar + phosphate); however, the right margin of the gap contains the baseless sugar-phosphate residue.
In order to fill the gap (replace the missing nucleotide), an enzyme specialized in synthesizing DNA, a DNA polymerase, will insert the correct nucleotide into the gap and link it to the normal nucleotide on the left margin by recognizing which base is opposite the gap on the complementary DNA strand. Figure 3, step 3 shows that the DNA polymerase recognizes that a G nucleotide is needed since the complementary base is a C. Note that an entire nucleotide is added here, not just a base. Before DNA polymerase is finished with the repair of the one-nucleotide gap, it removes the baseless sugar phosphate left behind by AP-endonuclease.
At this point, repair of the gap is almost, but not quite, finished, since there is a "nick" in the top DNA strand at the right margin of the former gap. Thus, the final step in the BER pathway is to ligate the DNA strands on both sides of the nick (Figure 3, step 4). If we examine the sugar phosphate DNA chain shown in Figure 2, we can see that the sugars that carry the DNA bases are linked together by phosphate groups. This type of linkage is referred to as a phosphodiester bond. The enzyme DNA ligase joins the strands by creating a phosphodiester bond between them, sealing the nick. In summary, the basic steps of the BER pathway are damage recognition and base excision, AP-site incision, DNA repair synthesis, and DNA ligation.
Nucleotide Excision Repair
DNA damage that involves particularly "bulky" molecules or chemical bonds between bases, or that significantly distorts the double-stranded structure of DNA, is subject to repair by the nucleotide excision repair (NER) pathway. For example, it has long been known that the ultraviolet (UV) light in sunshine can damage DNA by forming what are called photoproducts. UV radiation excites many types of molecules, causing them to react with each other and with DNA. In particular, UV light can catalyze the formation of chemical bonds between adjacent thymine and/or cytosine bases; these bonds are called intra-strand UV crosslinks (Figure 4A). These crosslinked bases distort the double-stranded structure of DNA and block DNA replication.
A second example of bulky DNA damage is that caused by large, organic molecules like aflatoxin, found in mold-contaminated peanuts, and benzo[ a ]pyrene (Figure 4B), a main component of smoke and soot. Both aflatoxin and benzo[ a ]pyrene are potent carcinogens. Ingestion or inhalation of these and similar compounds activates the body's detoxification systems, which convert the hydrophobic organic molecules into water-soluble forms for removal. However, the intermediate forms of aflatoxin and benzo[ a ]pyrene produced during the detoxification reaction happen to be very reactive with DNA purines, and form DNA base adducts (they "add on" to DNA). Specifically, such compounds tend to adduct guanine and, to a lesser extent, adenine. These large DNA adducts can cause mutations, and, since they block DNA replication, deletions of large segments of DNA can occur. Also, they activate the cell's damage surveillance systems, and, if not repaired, can cause cell death (apoptosis).
The mechanism of NER, involving some thirty proteins, is more complex than that of BER, but the basic principles are similar: damage recognition, damage excision, DNA repair synthesis, and DNA ligation (Figure 5). Damage recognition is obviously very important (Figure 5, step 1), but how can a single multiprotein complex detect so many different types of DNA damage? The answer is that the DNA damage must (1) distort the normal double-stranded structure of DNA, and/or (2) block transcription by RNA polymerase. Unusual kinks or twists in double-stranded DNA are recognized by the NER damage-recognition multiprotein complex. Also, when RNA polymerase stalls at a damaged DNA base, components of the NER damage-recognition complex are recruited to the site of damage.
Next, the double-stranded DNA adjacent to the damage is unwound by a DNA unwinding enzyme called a helicase (Figure 5, step 2). Unwinding of the DNA allows repair proteins access to the site of damage. The DNA strand containing the damaged base is then cleaved a few nucleotides after the damage, and about twenty-five nucleotides before it, by specific endonucleases associated with the NER protein complex (Figure 5, step 3). Endonucleases are enzymes that cleave inside a segment of DNA.
Next, the DNA segment that contains damage is displaced by DNA polymerase and associated proteins, and a corresponding repair patch is synthesized (Figure 5, step 4). Lastly, DNA ligase seals the nick, joining the newly synthesized piece of DNA to the preexisting strand (Figure 5, step 5).
Dna Mismatch Repair
The DNA mismatch repair (MMR) pathway has evolved to correct errors made by DNA polymerase during DNA replication. Such errors fall into two broad categories: base substitutions and insertions/deletions. A base substitution error occurs when DNA polymerase inserts an incorrect (noncomplementary) nucleotide opposite the template base, like a T opposite G instead of C, or A opposite C instead of G. These incorrect base pairs are referred to as mispairs or mismatches. Often, DNA polymerase will make a base substitution error when copying a base that has been damaged by alkylation. For example, DNA polymerase will frequently insert a T opposite O6-methylguanine on the other strand.
An insertion error occurs when DNA polymerase adds one or more extra nucleotides (+1, +2, +3, and so on) to a sequence; a deletion error is made when one or more nucleotides (−1, −2, −3, and so on) are omitted from a sequence. Sequences that contain repeats of the same nucleotide (mononucleotide repeat), such as AAAAAAAA, are particularly vulnerable to +1 or −1 insertion/deletion errors when copied by DNA polymerase. Such sequences might be called "slippery," in that DNA polymerase can "slide" on the DNA and lose its place. Other repetitive sequences, like the dinucleotide repeat CACACACA and the trinucleotide repeat CTGCTGCTG, are prone to +2 and +3, or −2 and −3 insertion/deletion errors, respectively. These repetitive DNA sequences are called microsatellites.
Defects in DNA mismatch repair have been found in several types of cancer, notably colon cancer, and microsatellite sequences that are either shorter or longer than normal are a hallmark of defective MMR. Expansion of trinucleotide repeat sequences is associated with a number of hereditary neurological disorders, such as fragile X syndrome, myotonic dystrophy, and Huntington's disease.
The process of MMR, like the BER and NER pathways, comprises damage recognition, damage excision, DNA repair synthesis, and DNA ligation. First, a mismatch or insertion/deletion error must be recognized by a complex of proteins specialized for the particular type of damage (mismatch, or small or large insertion/deletion). Just how the mismatch recognition protein complex "knows" which DNA strand contains the "right" nucleotide and/or which DNA strand contains the "wrong" one has not yet been determined.
Next, a phosphodiester bond in the DNA strand containing the mismatched nucleotide is cleaved by an endonuclease, the strand is displaced by DNA helicase, and a portion of the strand is removed by a combination of DNA exonuclease and DNA polymerase. Lastly, DNA polymerase carries out DNA repair synthesis, and DNA ligase restores the continuity of the sugar-phosphate-DNA backbone. The patch of DNA newly synthesized by the MMR DNA polymerase is relatively large, approximately 1,000 nucleotides long, compared to the DNA repair synthesis that takes place during BER, which typically replaces 1 nucleotide, or NER, which replaces approximately 30 nucleotides. MMR is especially important in tissues that are constantly regenerating, like the intestinal lining and the endometrium (the lining of the uterus), since growth requires DNA replication, which sometimes makes mistakes.
Future Directions
In addition to the three critical DNA repair pathways already discussed (BER, NER, and MMR), there are two additional types of DNA repair: double-strand break repair and recombinational repair. These are both complex phenomena, and scientists' understanding of them is still at an early stage. Also, many questions about BER, NER, and MMR still await answers. For example, since DNA damage that escapes repair leads to deleterious alterations of our DNA, could we prevent mutation by increasing the levels of DNA repair proteins? Could we live longer and healthier lives with more or better DNA repair? How are DNA repair pathways regulated by the cell? Is there such a thing as too much DNA repair? If repairs always took place whenever DNA damage occurred, would there be no evolution? Exactly how do the proteins and enzymes involved in DNA repair accomplish their jobs? These and many other exciting lines of inquiry are in store for future investigators.
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—Samuel E. Bennett and Dale Mosbaugh
