All cells have repair mechanisms that fi x DNA that has been damaged or has suffered errors during replication. Repair systems in bacteria that operate during DNA replication and cor-rect erroneous insertions of nucleotides are described next. Such errors cause relatively minor topological changes in the DNA.
Editing repair
Occasionally the wrong base is inserted in the copy strand (e.g., a T rather than a C opposite a G). This can occur if a base tautomerizes to a form that allows hydrogen bonding to the wrong partner. See Fig. 3.21 for an explanation. If the wrong nucleotide is added to the growing chain (e.g., a T opposite a G), the T must be removed;
otherwise, when the strand containing the G–T pair is replicated, one of its progeny duplexes will have an A–T pair in place of the G–C pair:
that is, a mutation will result. When the wrong base is inserted in the growing chain so that a mismatched pair results (e.g., G–T), DNA repli-cation stops: that is, the DNA polymerase does not continue to the next position. Presumably this occurs because of the resultant topological change in the DNA (distortion in the double-stranded helix). The incorrect nucleotide is then removed via a 3′-exonuclease. DNA poly-merases have 3′-exonuclease activity that func-tions in proofreading and removes mismatched nucleotides when they are added. Mutations in the dnaQ gene (mutD), which codes for a 3′-exonuclease subunit in the DNA polymerase III holoenzyme, result in greatly increased rates of spontaneous mutations.
Methyl-directed mismatch repair
The proofreading system is not perfect, and a certain number of wrong bases do get inserted into the newly replicated DNA. These can be removed, and the fi delity of DNA replication but not chromosomal replication, although
they can cause frameshift mutations. (See note 56 for an explanation.) If the concentration is suffi ciently high, chromosomal replication is inhibited. Chemicals that inhibit the synthesis of precursors to DNA include the monophos-phate of 5-fl uorodeoxyuridine, aminopterin, and methotrexate.
5-Fluorodeoxyuridine-5′-phosphate [FdUMP, made by cells from fl uorouracil (FU)] inhibits thymidylate synthase, which is the enzyme that converts dUMP to dTMP. (See Fig. 3.20.) In this reaction a methylene group (–CH2) and hydride (H:) are transferred from methylenetetra-hydrofolate (methylene–THF) to dUMP to form
Fig. 3.20 Inhibition of dTMP synthesis by anti-cancer drugs. 5-Fluorouracil is converted to 5-fl uorodeoxyuridine-5′-phosphate (fl uorodeoxyu-ridylate, or FdUMP), which inhibits thymidylate synthase (1), the enzyme that converts dUMP to dTMP. A methylene group (–CH2) and a hydride (H:) are transferred from methylenetetrahydrofolate (methylene-THF) to dUMP to form a methyl (–CH3), which converts dUMP to dTMP. As a consequence of losing the hydride, the THF is oxidized to dihy-drofolate. The dihydrofolate is reduced back to THF by NADPH in a reaction catalyzed by dihydrofolate reductase (2). The dihydrofolate reductase is inhib-ited by dihydrofolate analogues such as methotrex-ate and aminopterin.
region of DNA containing the GATC sequence.
Thus, for a few seconds or minutes after replica-tion, the template strand is fully methylated but the copy strand is undermethylated. During this brief period, the copy strand can be repaired.
What happens is that the copy strand with the incorrect base is cut at the 5′ side of the G in the unmethylated GATC and the newly synthesized DNA is removed by an exonuclease to a point just beyond the mismatch (Fig. 3.22). The gap is then fi lled with DNA polymerase III and sealed with DNA ligase.
Several proteins are involved in mismatch repair. In E. coli, these include the products of mutH, mutL, and mutS. MutH, MutL, and MutS are thought to form a complex with the DNA. In this complex, MutH is the endonu-clease. According to the model, MutS binds to the single base pair mismatch. Then MutL binds to MutS. MutH binds to a nearby GATC sequence, and its endonuclease activity is stimu-lated by the MutS/MutL complex. (Another mut gene, mutD, encodes the DNA polymerase III 3′ exonuclease, which is important for editing, as described earlier in the subsection entitled Editing repair.) DNA helicase II (the product of the mutU gene) is also required, and it is thought that its role is to unwind the cut strand so that it can be degraded by exonuclease.
More than one exonuclease can be used. If the endonucleolytic cut by MutH is made 3′ to the mismatch, then exonuclease I (ExoI) or ExoX, which is a 3′- to 5′-exonuclease is used.
If the cut is made 5′ to the mismatch, then either exonuclease VII (ExoVII) or RecJ, which are 5′- to 3′-exonucleases, are used. DNA poly-merase III fi lls in the gap, and DNA ligase seals the gap.
Mismatch repair can also be used to repair DNA damaged by the incorporation of base analogues or by certain types of alkylating agent as long as the distortion of the double helix is not severe. Otherwise, repair mechanisms described in Section 19.2.1 are used. Null muta-tions in any of the mut genes involved in mis-match repair result in a 102- to 103-fold increase in mutation frequency in E. coli. Homologues of the mut genes can be found in eukaryotes such as yeast, mice, and humans, where loss of function also results in increased mutation rates and, in the case of humans, is correlated with cancer in tumor cell lines from certain tissues improved 102- to 103-fold (Fig. 3.22), by what
is called the methyl-directed mismatch repair (MMR) system (reviewed in refs. 57 and 58).
The nucleotide that is removed is the incorrect nucleotide in the copy strand rather than the template strand; thus the template strand is not changed, and a mutation does not occur.
The repair system can distinguish the copy strand from the template strand because the template strand is marked by methylation.
E. coli has an enzyme called deoxyadenosine methylase (Dam methylase) that methylates all adenines at the N6 position within 5′-GATC-3′
sequences. (The sequence is a palindrome and therefore is present in both strands but in oppo-site orientation, i.e., 3′-CTAG-5′.) However, the enzyme does not begin to methylate the DNA until a short period after replication of the Fig. 3.21 Tautomerization can lead to incorrect base pairing. Two isomers in equilibrium that differ in the arrangements of their atoms are called tautomers.
Commonly, tautomers differ in the placement of hydrogen. For example, one isomer might exist in the enol form (–OH attached to a carbon–carbon double bond), whereas the other isomer might exist in the keto form (contains a C=O group). Keto–enol tautomerizations greatly favor the keto form. When thymine tautomerizes from the keto to the enol form, a proton dissociates from the nitrogen in the ring and moves to the oxygen in the keto group to form the hydroxyl. When this happens, two electrons shift in from the nitrogen to form the C=N. (A) Adenine (A) correctly base pairs with the keto form of thymine (T). (B) Thymine has tautomerized to the enol form and base pairs with guanine (G) to form a mismatch.
(e.g., colorectal carcinomas). (For reviews, see refs. 59–61.)
3.2 Summary
To replicate DNA, several problems must be attended to. These include unwinding the double helix without causing it to overwind
in a positive supercoil in the unreplicated por-tion, creating a primer that can be extended by DNA polymerase III, and keeping the poly-merase that synthesizes the lagging strand at the replication fork. These and other factors necessitate using about 30 different proteins to replicate the DNA. The replication fork is cre-ated at a specifi c sequence in the DNA (called Fig. 3.22 Mismatch repair can occur when the wrong nucleotide has been inserted. For example, a T instead of a C might be inserted opposite a G. The template strand is methylated at an adenine in a CTAGsequence.
The newly synthesized strand is not yet methylated, and this aids the repair enzymes in distin-guishing between the newly synthesized strand and the template strand. (There is a slight delay in the methy-lation of newly syn-thesized DNA. However, the A in GATC eventually becomes methylated.) (A) Anendonucleolytic cut is made either on the 5′side or the 3′side of the mismatch in the GATC sequence in thenonmethylated strand. (B) An exonuclease then removes the newly synthesized DNA past the point of themismatch. This requires helicase II (MutU). (Helicase II is not identical to DnaB, which unwinds the strandsduring DNA replication.) If the mismatch is on the 5′side of the cut, then exonuclease I degrades the DNA 3′to 5′through the mismatch. If the mismatch is on the 3′side of the cut, then exonuclease VII or RecJ proteindegrades the DNA 5′to 3′through the mismatch. (C) DNA polymerase III then fills in the missing DNA, andthe gap is sealed with DNA ligase.
Single-stranded DNA-binding protein is also required. The proteinsinvolved in recognizing the mismatch and making the cuts are MutH, MutS, and MutL. The MutS proteinrecognizes the mismatch. The MutH protein is the endonuclease that makes the cut. It recognizes the GATCsequence and cleaves the unmethylated DNA on the 5′side of the G in the GATC. MutS and MutH form acomplex in which they are linked by MutL.
Muk, Par, SetB, and MreB proteins. The MreB proteins are related to actin.
Errors made during replication are repaired in two ways. Editing repair involves the imme-diate removal of the incorrect nucleotide by the 3′-exonuclease activity of DNA polymerase III.
The second method of repair, called mismatch repair, can occur if the polymerase fails to remove the incorrect nucleotide and proceeds beyond the error site. During mismatch repair an exonuclease removes the segment of copy DNA that includes the mismatched nucleotide pair, and the gap is fi lled with DNA polymerase III and sealed with DNA ligase. The copy strand is recognized because it is undermethylated for a short period after synthesis.
Study Questions
1. Describe the roles of the following proteins