Textbook Assignment: Chapter 5, Pages 142 - 155 (includes end-of-chapter material for entire chapter).
Major concepts
Introduction: The notes for this lecture, which are adapted from those used with the previous textbook for this course, tend to focus more on thymine dimers as model lesions to be removed from damaged DNA than our current textbook does. Most of the repair systems are effective against a variety of spontaneous or induced abnormalities in the DNA double helix. Thus, the examples in these notes are appropriate for the repair systems discussed even though the current textbook takes a slightly different approach.
Thymine dimers: Ultraviolet radiation causes a dimerization reaction between adjacent thymine residues in DNA (fig. 5.11) that disrupts normal base pairing and normal DNA function in the immediate area of the dimers. A variety of mechanisms exist to reverse such damage in all types of cells, ranging from bacteria to higher eukaryotes. Many of the same mechanisms are also used to repair other types of DNA damage that lead to mismatches of base pairing or single-stranded gaps. One of the repair mechanisms is the so-called SOS response, discussed later in this lecture, which results in error-prone repair and thus produces mutations.
Photoreactivation: The first line of defense against uv damage in bacterial cells is photoreactivation. In E. coli an enzyme produced from the phr locus, called photolyase (in our textbook called photoreactivation enzyme, PRE), binds to the thymine dimers in the dark and then uses visible light energy to break the dimer bonds, restoring the original sequence of normal bases (TT). This directly reverses the dimerization process without any need for excision of the damage and replacement of the missing bases (fig. 5.18). Similar photoreactivation enzymes have been widely conserved across the evolution of highly diverse species.
Excision repair systems: In cell strains that lack the photoreactivation system, and in other cases where it has failed to repair the damage, a variety of excision repair systems can be called into action. Such systems remove the immediate damage and some amount of the strand that contained it, followed by synthesis of a new strand, using the undamaged partner as a template.
Excision of ultraviolet damage: In E. coli, a special endonuclease consisting of three subunits produced from the uvr-A, uvr-B, and uvr-C genes nicks the damaged strand at specific locations upstream and downstream from a thymine dimer. This allows the damaged segment to be removed, leaving a gap that is then filled in by DNA polymerase I and closed by DNA ligase. There is also a similar, but somewhat more complex excision repair system in eukaryoic cells.
AP repair: Enzymes known as DNA glycosylases are capable of removing damaged bases from DNA by hydrolyzing the bond between the base and the 1'-position of deoxyribose. This leaves apurinic or apyrimidinic sites in the double stranded DNA. Specific AP endonucleases then nick the damaged strand upstream and downstream from the AP site, resulting in removal of a short segment, which is then replaced by DNA polymerase I and DNA ligase in a manner similar to excision repair.
Proofreading and mismatch repair: This system was described in greater detail in the previous text for this course (Page 411, Klug and Cummings, available from Norlin reserve). I have chosen to retain some of that detail in these notes even though it is not covered in our current text. When a mismatch is detected in a newly synthesized segment of DNA, a mismatch repair system is called into action.
Which strand contains the error? The first problem is for the system to decide which is the parental (correct) strand and which is the newly synthesized (presumably error-containing) strand. Methylation of the adenine in GATC sequences serves temporarily as a marker for the parental strand in newly synthesized DNA in E. coli. GATC forms a pallindromic sequence in double stranded DNA (one that reads the same in either direction). If one starts with GATC methylated in both strands, the newly synthesized complementary strands will temporarily lack methylation. GATC sequences with one adenine methylated and the other not are recognized by an enzyme known as deoxyadenosine methylase (dam), which methylates the new strand. However, there is a short time lag after replication before this occurs. During those few minutes, the parental strand is methylated and the newly-synthesized strand is not.
Mismatch detection: Four proteins, coded by genes designated mutH, mutL, mutS, and mutU, participate in mismatch repair. The S and L proteins are involved in recognition of the mismatch (which distorts the normal double helical structure). The H protein recognizes the newly synthesized strand (not methylated) and nicks it. The U protein helps to open the nicked area, which is then stabilized in the open configuration by single-stranded DNA-binding protein. DNA polymerase I and ligase then make the repair, using the methylated strand as a template. The whole process requires about three minutes in E. coli. This proofreading system catches errors missed by the proofreading function of DNA polymerase III, and is believed to improve fidelity of DNA synthesis by about two orders of magnitude (100 fold).
Postreplicative repair: If damage such as a thymine dimer is not detected and removed, it will cause problems during the next round of DNA synthesis that can result in its repair. When DNA polymerase III encounters a thymine dimer or similar lesion, it temporarily stops synthesis, leaving a gap in the newly synthesized strand, which is started again further along the template strand that contains the dimer. This gap triggers a process known as postreplicative repair or recombinational repair (fig. 5.20). A protein known as the RecA protein (coded at the recA locus) coats the single stranded DNA (the template strand that failed to make a partner because it contains a lesion). This coated strand then invades the newly replicated double-stranded DNA that has just been synthesized from the other template strand at the same replication fork, and causes the damaged strand (with the dimer) to pair with the complementary undamaged template strand. This leaves the newly synthesized undamaged strand unpaired, which is not a problem because it is a correct template for repair enzymes to replace the strand that has been taken from it and paired with the damaged strand. The damaged strand and its newly found undamaged partner now have another chance for any of the possible repair mechanisms to remove the lesion before the next round of replication. This repair system is one component of a complex response of bacterial cells to a signal of distress (DNA damage). The overall response, which is summarized briefly below, is known as the SOS response.
The SOS response: In addition to the recombinational repair described above, interaction of the recA protein with single stranded DNA (indicative of genetic damage the cell is attempting to repair) causes the recA protein to gain the ability to inactivate a regulatory protein known as LexA. The normal function of the LexA protein is to repress expression of about 20 genes that are involved in various types of emergency repair processes (fig. 5.21). These include some genes whose products stop the cell cycle, giving the cell more time to achieve appropriate repairs when an emergency is declared (by the absence of enough LexA to keep the emergency response proteins turned off). Each of the genes that is repressed by LexA has a consensus sequence in its promoter, called the SOS box (5'-CTGX10CAG-3') that serves as a binding site for the LexA protein. Among the genes that are activated are those involved in excision of thymine dimers, as well as a variety of others. In addition to triggering various normal repair syystems, RecA and two proteins whose expression is activated by it, UmuC and UmuD, allow DNA synthesis to proceed across the lesion in an error-prone fashion (fig. 5.22). In many cases, the cell is better off replicating its DNA with errors (which may not disrupt essential functions) than not being able to replicate its DNA at all.
Transcription-Repair Coupling: DNA that is being actively transcribed is more likely to be reapired than DNA that is not. If prokaryotic RNA polymerase encounters a lesion in the template strand that stops transcription, a transcription-repair coupling factor (TRCF) binds to the stalled RNA polymerase, removes the polymerase and releases the partial transcript, which is then degraded. The binding of TRCF also activates an excision-repair system (fig. 5.23). In eukaryotic cells (fig. 5.24), the overall process is similar except the polymerase remains attached and resumes transcription after the repair has been achieved. Only the template (antisense) strand is repaired by transcription-coupled mechanisms (boxed example 5.4).
Loss of repair systems: Mutations in the genes that code for components of DNA repair systems can greatly increase the sensitivity of cells or organisms to mutation. Bacterial and yeast strains that have been extensively mutated to disable their repair systems are often employed as highly sensitive detection systems for mutagenic (and carcinogenic) chemicals. These include the well-known Ames test for carcinogenic activity, which is based on mutational analysis in repair-deficient bacteria.
Human diseases caused by loss of DNA repair systems: DNA repair systems play a major role in normal human health. Two examples of human pathology caused by loss of repair systems are described below.
Xeroderma pigmentosum: Xeroderma pigmentosum is a human genetic disease (or more correctly, a family of closely related genetic diseases), in which there is abnormal sensitivity to ultraviolet radiation. A number of different genes appear to be involved. Some patients exhibit defects in photoreactivation, but loss of excision repair is more common. Mutations in at least seven different genes coding for proteins involved in excision repair can cause afflicted individuals to exhibit the symptoms of xeroderma pigmentosum.
Cockayne syndrome: This human genetic disease, whose symptoms include mental retardation, dwarfism, and premature aging, appears to be primarily due to failure of transcription-repair coupling.