BCH 5045 Graduate Survey of Biochemistryhort.ifas.ufl.edu/faculty/guy/bch5045/Lecture Files/Lecture 25.pdf · FIGURE 25-13 Synthesis of Okazaki fragments. \⠀愀尩 At intervals,

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BCH 5045

Graduate Survey of Biochemistry

Instructor: Charles Guy Producer: Ron Thomas

Director: Marsha Durosier

Lecture 25 Slide sets available at:

http://hort.ifas.ufl.edu/teach/guyweb/bch5045/index.html

http://hort.ifas.ufl.edu/teach/guyweb/bch5045/index.html�



• LEHNINGER • PRINCIPLES OF BIOCHEMISTRY

• Fifth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 25 DNA Metabolism



The enzyme primase synthesizes an RNA primer for a new Okazaki fragment synthesis to begin.

Presenter

Presentation Notes

FIGURE 25-13 Synthesis of Okazaki fragments. (a) At intervals, primase synthesizes an RNA primer for a new Okazaki fragment. Note that if we consider the two template strands as lying side by side, lagging strand synthesis formally proceeds in the opposite direction from fork movement. (b) Each primer is extended by DNA polymerase III. (c) DNA synthesis continues until the fragment extends as far as the primer of the previously added Okazaki fragment. A new primer is synthesized near the replication fork to begin the process again.



Overall process of DNA synthesis on the leading and lagging strands.

Presenter

Presentation Notes

FIGURE 25-14 DNA synthesis on the leading and lagging strands. Events at the replication fork are coordinated by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. This figure shows the replication process already underway (parts (a) through (e) are discussed in the text). The lagging strand is looped so that DNA synthesis proceeds steadily on both the leading and lagging strand templates at the same time. Red arrows indicate the 3′ end of the two new strands and the direction of DNA synthesis. The heavy black arrows show the direction of movement of the parent DNA through the complex. An Okazaki fragment is being synthesized on the lagging strand. The subunit colors and the functions of the clamp-loading complex are explained in Figure 25-15.



The function of type II topoisomerase in replication termination and separation of catenated interwound chromosomes.

Presenter

Presentation Notes

FIGURE 25-19 Role of topoisomerases in replication termination. Replication of the DNA separating opposing replication forks leaves the completed chromosomes joined as catenanes, or topologically interlinked circles. The circles are not covalently linked, but because they are interwound and each is covalently closed, they cannot be separated—except by the action of topoisomerases. In E. coli, a type II topoisomerase known as DNA topoisomerase IV plays the primary role in the separation of catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break.



Eukaryotic replication origin. Is there just one replication origin per chromosome in humans? Exactly how is replication of eukaryotic chromosomes accomplished?

Presenter

Presentation Notes

FIGURE 25-20 Assembly of a pre-replicative complex at a eukaryotic replication origin. The initiation site (origin) is bound by ORC, CDC6, and CDT1. These proteins, many of them AAA+ ATPases, promote loading of the replicative helicase, MCM2–7, in a reaction that is analogous to the loading of the bacterial DnaB helicase by DnaC protein. Loading of the MCM helicase complex onto the DNA forms the pre-replicative complex, or pre-RC, and is the key step in the initiation of replication.



The Ames test for potential carcinogens is based on mutagenic effects of compounds on a strain of Salmonella typhimurium having a mutation that inactivates an enzyme of the histidine biosynthetic pathway so that the cells can’t make enough histidine when plated on a histidine-free medium. Few cells grow. If a compound is mutagenic, some of the mutations restore the ability to make histidine. Of b, c and d, which is the most mutagenic and the least toxic?

Test compound in filter disk

Presenter

Presentation Notes

FIGURE 25-21 Ames test for carcinogens, based on their mutagenicity. A strain of Salmonella typhimurium having a mutation that inactivates an enzyme of the histidine biosynthetic pathway is plated on a histidine-free medium. Few cells grow. (a) The few small colonies of S. typhimurium that do grow on a histidine-free medium carry spontaneous back-mutations that permit the histidine biosynthetic pathway to operate. Three identical nutrient plates (b), (c), and (d) have been inoculated with an equal number of cells. Each plate then receives a disk of filter paper containing progressively lower concentrations of a mutagen. The mutagen greatly increases the rate of back-mutation and hence the number of colonies. The clear areas around the filter paper indicate where the concentration of mutagen is so high that it is lethal to the cells. As the mutagen diffuses away from the filter paper, it is diluted to sublethal concentrations that promote back-mutation. Mutagens can be compared on the basis of their effect on mutation rate. Because many compounds undergo a variety of chemical transformations after entering cells, compounds are sometimes tested for mutagenicity after first incubating them with a liver extract. Some substances have been found to be mutagenic only after this treatment.



DNA methylation of individual strands can serve to distinguish parent (template) strands from newly synthesized strands.

Presenter

Presentation Notes

TABLE 25-5 Types of DNA Repair Systems in E. coli. FIGURE 25-22 Methylation and mismatch repair. Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strands in E. coli DNA, a function that is critical to mismatch repair (see Figure 25-23). The methylation occurs at the N6 of adenines in (5′)GATC sequences. This sequence is a palindrome (see Figure 8-18), present in opposite orientations on the two strands.



Location of cleavage site relative to the mismatch pair results in one of two alternative pathways of repair.

Presenter

Presentation Notes

FIGURE 25-23 A model for the early steps of methyl-directed mismatch repair. The proteins involved in this process in E. coli have been purified (see Table 25-5). Recognition of the sequence (5′)GATC and of the mismatch are specialized functions of the MutH and MutS proteins, respectively. The MutL protein forms a complex with MutS at the mismatch. DNA is threaded through this complex such that the complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at a hemimethylated GATC sequence. MutH cleaves the unmethylated strand on the 5′ side of the G in this sequence. A complex consisting of DNA helicase II and one of several exonucleases then degrades the unmethylated DNA strand from that point toward the mismatch (see Figure 25-24). FIGURE 25-24 Completing methyl-directed mismatch repair. The combined action of DNA helicase II, SSB, and one of four different exonucleases removes a segment of the new strand between the MutH cleavage site and a point just beyond the mismatch. The exonuclease that is used depends on the location of the cleavage site relative to the mismatch, as shown by the alternative pathways here. The resulting gap is filled in (dashed line) by DNA polymerase III, and the nick is sealed by DNA ligase (not shown).



DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone.

deoxyribose

Presenter

Presentation Notes

FIGURE 25-25 DNA repair by the base-excision repair pathway. 1 A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone. 2 An AP endonuclease cleaves the phosphodiester backbone near the AP site. 3 DNA polymerase I initiates repair synthesis from the free 3′ hydroxyl at the nick, removing (with its 5′→3′ exonuclease activity) and replacing a portion of the damaged strand. 4 The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.



Nucleotide-excision repair is similar in all organisms.

E. coli humans

Presenter

Presentation Notes

FIGURE 25-26 Nucleotide-excision repair in E. coli and humans. The general pathway of nucleotide-excision repair is similar in all organisms. 1 An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion. 2 The DNA segment—of 13 nucleotides (13 mer) or 29 nucleotides (29 mer)—is removed with the aid of a helicase. 3 The gap is filled in by DNA polymerase, and 4 the remaining nick is sealed with DNA ligase.



Photolyase repair of pyrimidine dimers. Energy from absorbed light is used to reverse the photoreaction lesion. No blue light no repair.

Presenter

Presentation Notes

MECHANISM FIGURE 25-27 Repair of pyrimidine dimers with photolyase. Energy derived from absorbed light is used to reverse the photoreaction that caused the lesion. The two chromophores in E. coli photolyase (Mr 54,000), N5,N10-methenyltetrahydrofolylpolyglutamate (MTHFpolyGlu) and FADH–, perform complementary functions. MTHFpolyGlu functions as a photoantenna to absorb blue-light photons. The excitation energy passes to FADH–, and the excited flavin (*FADH–) donates an electron to the pyrimidine dimer, leading to the rearrangement as shown



Why does the mistaken O6-methylation of guanine give rise to G-C pair to an A-T pair?

Presenter

Presentation Notes

FIGURE 25-28a Example of how DNA damage results in mutations. (a) The methylation product O6-methylguanine pairs with thymine rather than cytosine. FIGURE 25-28b Example of how DNA damage results in mutations. (b) If not repaired, this leads to a G≡C to A=T mutation after replication.



Presenter

Presentation Notes

TABLE 25-6 Genes Induced as Part of the SOS Response in E. coli



Near perfect DNA replication is critical to prevent the accumulation of mutations, some of which would be cancer-causing in humans. Genetic disorders such as Fanconi's anemia (FA) and Bloom's syndrome (BS) are considered the result of defects in replication and DNA repair. Both syndromes are autosomal recessive disorders. Exposure to hydroxyurea (HU) is known to damage DNA and stall replication (what kind of agent is HU?). FA causes cells to be extra sensitive to agents that induce damaging DNA interstrand crosslinks and chromosomal instability, and thus the disease is thought to result from a defect in DNA repair. Fanconi's anemia occurs when a person receives one copy of an abnormal gene from each parent. The disease is often diagnosed in children between 2 and 15 years old. With Fanconi's anemia, people have low numbers of white and red blood cells, and platelets. Other, but not all symptoms include abnormal heart, lungs, and digestive tract, bone problems, and dark areas of the skin called “Cafe au lait spots.” People with a mild form of Fanconi's anemia and with mild to moderate blood cell changes may not need transfusions, but they do need regular check-ups and blood count checks and close monitoring for the development of several types of blood disorders and cancers, often leukemia, as well as cancers of the head, neck, or urinary system. More than a dozen genes for have been associated with FA. One gene linked to FA is the helicase FANCJ, and FANCJ-deficient cells are sensitive to HU. Recent research has shown that FANCJ and another helicase, BS helicase (BLM) which is associated with Bloom’s syndrome, appear to cooperate in response to DNA damage during replication. FANCJ and BLM helicases appear to work together in a process where the two proteins are able to unwind forked DNA substrates that is a reflection of an increased efficiency to unwind damaged DNA which would be important in DNA repair and chromosomal stability.

This slide is not in the lecture video

(Suhasini et al., (2011) EMBO J. 30, 692–705)

Documents

BCH 5045 Graduate Survey of Biochemistryhort.ifas.ufl.edu/faculty/guy/bch5045/Lecture Files/Lecture 25.pdf · FIGURE 25-13 Synthesis of Okazaki fragments. \⠀愀尩 At intervals,