Garrett and Grisham, Biochemistry, Third Edition DNA Metabolism: Replication, Recombination, and...

Garrett and Grisham, Biochemistry, Third Edition

DNA Metabolism: Replication, Recombination, and Repair

Chapter 28

Biochemistryby

Reginald Garrett and Charles Grisham

Igor ChesnokovDepartment of Biochemistry and Molecular GeneticsOffice Phone # 934-6974E-mail: [email protected]


Outline• How Is DNA Replicated?• What Are the Properties of DNA Polymerases?• How Is DNA Replicated in Eukaryotic Cells?• How Are the Ends of Chromosomes Replicated?• How Are RNA Genomes Replicated?• How Is the Genetic Information Shuffled by Genetic

Recombination?• Can DNA Be Repaired?• What Is the Molecular Basis of Mutation?


How Are RNA Genomes Replicated?

• Many viruses have genomes composed of RNA

• Can viral RNA serve as a template for DNA synthesis?

• What enzyme could mediate such process?


Another Way to Make DNA

RNA-Directed DNA Polymerase • 1964: Howard Temin notices that DNA

synthesis inhibitors prevent infection of cells in culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor virus replication

• 1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka "reverse trascriptase"


Reverse Transcriptase

• All RNA tumor viruses contain a reverse transcriptase

• Unusual primer is required - a tRNA molecule that the virus captures from the host

• RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid


Reverse Transcriptase Activities • Three enzyme activities

– RNA-directed DNA polymerase– RNase H activity - degrades RNA in the

DNA:RNA hybrids– DNA-directed DNA polymerase - which

makes a DNA duplex after RNase H activity destroys the viral genome

The structures of AZT (3¢-azido-2¢,3¢-dideoxythymidine).

This nucleoside was the first approved drug for treatment of AIDS. AZT is phosphorylated in vivo to give AZTTP (AZT 5¢-triphosphate), a substrate analog that binds to HIV reverse transcriptase, HIV reverse transcriptase incorporates AZTTP into growing DNA chains in place of dTTP. Incorporated AZTMP blocks further chain elongation because its 3¢-azido group cannot form a phosphodiester bond with an incoming nucleotide.

Host cell DNA polymerases have little affinity for AZTTP.


Genetic recombination;major types

• Homologous recombination involves similar DNA sequences

• Non-homologous recombination – when very different nucleotide sequences recombine, occurs at low frequency

• Transposition – enzymatic insertion of transposon (mobile segment of DNA)

• Nonhomologous recombination and transposition play significant evolutionary role


Homologous recombination

• Recombination involving similar DNA sequences is called homologous recombination

• Homologous recombination is achieved by the process of general recombination

• General recombination requires the breakage and reunion of DNA strands

• The proteins responsible include RecA, RecBCD, RuvA, RuvB, & RuvC

Meselson and Weigle’s experiment demonstrated that a physical exchange of chromosome parts actually occurs during recombination.

Density-labeled, “heavy” phage, (ABC), were used to co-infect bacteria along with ”light” (abc) phage. The progeny from the infection were collected and subjected to CsCl density gradient centrifugation.

Parental-type heavy (ABC) and light (abc) phage were well separated in the gradient, but recombinant phage particles (ABc,Abc, aBc,aBC, and so on ) were distributed diffusely between the two parental bands because they contained chromosomes constituted from fragments of both “heavy” and “light” DNA.

These recombinant chromosomes formed by breakage and reunion of parental “heavy” and “light” chromosomes.


Mechanism of Recombination• Any pair of homologous DNA segments

can be used as substrates• In 1964, Robin Holliday proposed a

model involving single-stranded nicks at homologous sites

• Duplex unwinding, strand invasion and ligation create a Holliday junction

The Holliday model for homologous recombination.

(A) Two homologous DNA duplexes are aligned – synapsis.

(B) Recombination begins with the introduction of single-stranded nicks at homologous sites on two chromosomes

(C) Strand invasion occurs through partial unwinding and base-pairing with the intact strand in the other duplex

(D) Free ends from different duplexes are ligated resulting in cross-stranded intermediate – Holliday junction

(E) Branches can migrate by unwinding and rewinding of two duplexes


The Holliday model for homologous recombination.

Branch migration (E) results in strand exchange. Another pair of nicks must be introduced to resolve Holliday junction into two DNA duplex molecules. Nicks take place either at E an W (- strands) or at N and S (+ strands) resulting in “patch” or “splice” recombinant heteroduplexes.


Enzymology of Recombination

• RecBCD initiates recombination in E.coli• RecA forms nucleoprotein filament for

strand invasion and homologous pairing• RuvA, RuvB, RuvC drive branch migration

and help to resolve the Holliday junction into recombination products

• Eukaryotic systems are probably similar, homologous proteins are identified in eukaryotes.


Model of RecBCD-dependent initiation of recombination.

RecBCD consists of three subunits and has both helicase and nuclease activities.

c site – recombinational “hotspot” (5-GCTGGTGG-3), more than 1000 in E.coli.



(a) RecBCD binds to a duplex DNA end, and its helicase activity begins to unwind the DNA double helix. “Rabbit ears” of ssDNA loop out from RecBCD because the rate of DNA unwinding exceeds the rate of ssDNA release by RecBCD.



(b) As it unwinds the DNA, SSB ( and some RecA) bind to the single-stranded regions; the RecBCD endonuclease activity randomly cleaves the ssDNA, showing a greater tendency to cut the 3’-terminal strand rather that the 5’-terminal strand.



(c) When RecBCD encounters a properly oriented c site, the 3¢-terminal strand is cleaved just below the 3¢-end of c.



(d) RecBCD now directs the binding of RecA to the 3¢-terminal strand, as RecBCD endonuclease activity now acts more often on the 5¢-terminal strand. (e) A nucleoprotein filament consisting of RecA-coated 3’-strand ssDNA is formed. This nucleoprotein filament is capable of homologous pairing with a dsDNA and strand invasion.


The RecA Protein – recombinase.

• 38 kD enzyme that catalyzes ATP-dependent DNA strand exchange, leading to formation of Holliday junction

• RecA forms a helical filament with a groove to accommodate DNA

• RecA:ssDNA complex binds dsDNA at secondary site and searches for regions homologous with the bound ssDNA, then forms the desired duplex

The structure of RecA, a 352-residue, 38-kD protein.

(a) Ribbon diagram of the RecA monomer. Note the ADP bound at the site near helices C and D.

(b) (b) RecA filament. Four turns of a helical filament that has six RecA monomers per turn. A RecA monomer is highlighted in red. RecA filament can bind multiple DNA strands!

Model for homologous recombination as promoted by RecA enzyme.

(a) RecA protein (and SSB) aid strand invasion of the 3’-ssDNA into a homologous DNA duplex,

(b) forming a D-loop.

(c) The D-loop strand, that has been displaced by strand invasion, pairs with its complementary strand in the original duplex to form a Holiday junction as strand invasion continues.


Resolving Holliday Junctions• Ruv proteins resolve the junction into

recombination products• RuvA and RuvB act as a helicase that

dissociates the RecA filament and catalyzes branch migration

• RuvC is an endonuclease that binds at the junction and cuts pairs of DNA strands of similar polarity. Both splice and patch recombinants can be produced.

Model for resolving Holliday junction.

(left) RuvA tetramer fits snugly within the Holliday junction point.

(center) RuvB hexameric rings assemble on opposite sides of DNA heteroduplexes and act as motors to promote branch migration by driving the passage of the DNA duplexes through themselves.

(right) RuvC resolvase binds to the Holliday junction and cuts it by its nuclease activity.


Knockout Mice: A Method to Investigate the Essentiality of a Gene

based on homologous recombination.


Transposons• In 1950, Barbara McClintock showed

that activator genes in corn could move freely about the genome.

• This was at first viewed as heresy• Molecular biologists in the late 1970s

confirmed what McClintock discovered• She received a MacArthur Award in

1981 and a Nobel Prize in 1983

The typical transposon has inverted nucleotide-sequence repeats at its termini, represented here as the 12-bp sequence ACGTACGTACGT (a). Transposon acts at a target sequence (shown here as the sequence CATGC) within host DNA by creating a staggered cut (b) whose protruding single-stranded ends are then ligated to the transposon (c). The gaps at the target site are then filled in, and the filled-in strands are ligated (d). Transposon insertion thus generates direct repeats of the target site in the host DNA, and these direct repeats flank the inserted transposon.


Can DNA Be Repaired?

A fundamental difference from RNA, protein or lipid• All the others can be replaced, but DNA must be

preserved • Cells require a means for repair of missing, altered

or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links

• The human genome has about 150 genes associated with DNA repair

• DNA repair systems include: direct reversal damage repair, single-strand damage repair, double strand break repair, and translesion DNA synthesis


DNA repair systems

• Double-strand breaks (DSBs) are a particular threat to genome stability, because lost sequence information cannot be recovered from the same DNA

• Chemical reactions that reverse the damage, returning DNA to its proper state, are direct reversal repair systems.

• Single-strand damage repair relies on the intact complementary strand to guide repair

• Systems repairing single-strand breaks include:– Mismatch repair (MMR)– Base excision repair (BER)– Nucleotide excision repair (NER)


Can DNA Be Repaired?DSB repair through nonhomologous DNA end joining (NHEJ). Ku70/80 binds the ends and recruits a set of proteins that juxtaposes the broken ends. Processing of the ends to generate proper substrates for DNA ligase IV then occurs, followed by DNA-ligase-mediated end joining.Double-strand breaks that arise during the S phase of the cell cycle can be repaired through homologous recombination.


Can DNA Be Repaired?DSB repair through homologous DNA recombination. The orange-red pair of lines symbolizes the double-stranded DNA with a DSB; the black-blue pair represents the sister chromatid. Homologous recombination creates a D-loop (c), and sister chromatid-directed DNA replication restores the information content of the damaged duplex (d-f). Depending on how the Holliday junctions are resolved, the products (g) are either (left) non-crossover or (right) crossover recombinants.


Can DNA Be Repaired?Restarting a stalled replication fork through homologous DNA recombination. A lesion in the DNA is symbolized by a circle; in this case, the lesion is in the leading-strand template (a). Leading-strand synthesis halts because of the lesion (b). Lagging-strand synthesis (red) continues, and the Okazaki fragments are ligated (c). When the leading strand invades the new DNA duplex formed by lagging-strand synthesis, a D-loop is formed and strand exchange occurs. Using the lagging strand as a template, synthesis of the leading strand (black) resumes (d), and the replication fork is reestablished (e).


Mismatch Repair corrects errors introduced during DNA replication

• Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it

• Example is a Methyl-directed pathway of E. coli • Since methylation occurs post-replication,

repair proteins (MutS, MutH, MutL) identify methylated strand as parent, remove mismatched bases on the other strand and replace them

• http://en.wikipedia.org/wiki/Mismatch_repair


Reversing Chemical Damage (excision repair)

• Pyrimidine dimers can be repaired by photolyase

• Excision repair: DNA glycosylase removes damaged base, creating an "AP site"

• AP endonuclease cleaves backbone, exonuclease removes several residues and gap is repaired by DNA polymerase and DNA ligase

UV irradiation causes dimerization of adjacent thymine bases. A cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the presence of such dimers. Photolyase can break cyclobutyl ring.

Base excision repair.

A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an apurinic acid (AP) site. Then, an apurinic/apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.


What Is the Molecular Basis of Mutation?

• Point mutations arise by inappropriate base-pairing

• Mutations can be caused by base analogs• Chemical mutagens react with bases in

DNA• Insertions and deletions result in frameshift

mutations


Types of mutationsLarge chromosomal deletionsTranslocations (chromosome segments

swapped)Point mutations:-Transition - Pu-Pu or Py-Py-Transversion - Py-Pu or Pu-Py

-Mis-sense 1 base for another

-FrameshiftAlters the reading frame of the genemust be in protein coding regioninsertion or deletion

Examples of point mutations due to base mispairings in unusual circumstances. (A) The rare imino tautomer of adenine base pairs cytosine rather than thymine. (1) The normal A-T base pair. (2) The A*-C base pair is possible for the adenine tautomer in which a proton has been transferred from the 6-NH2 of adenine to N-1. (3) Pairing of C with the imino tautomer of A (A*) leads to a transition mutation (A-T to G-C) appearing in the next generation. (B) A in the syn conformation pairing with G (G is in the usual anti conformation). (C) T and C form a base pair by H-bonding interactions mediated by a water molecule.

(a) 2-Aminopurine (adenine analog) normally base-pairs with T but (b) may also pair with cytosine through a single hydrogen bond.

Another example of base analogs. Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an A-T to G-C transition.

Examples of Chemical mutagens. (a) HNO2 (nitrous acid) converts cytosine to uracil and adenine to hypoxanthine. (b) Nitrosoamines, organic compounds that react to form nitrous acid, also lead to the oxidative deamination of A and C. (c) Hydroxylamine (NH2OH) reacts with cytosine, converting it to a derivative that base-pairs with adenine instead of guanine. The result is a C-G to T-A transition. (d) Alkylation of G residues to give methylguanine, which base-pairs with T. (e) Alkylating agents include nitrosoamines, nitrosoguanidines, nitrosoureas, alkyl sulfates, and nitrogen mustards.

Note that nitrosoamines are mutagenic in two ways: They can react to yield HNO2, or they can act as alkylating agents. The nitrosoguanidine, is a very potent mutagen used in laboratories to induce mutations in experimental organisms such as Drosophila melanogaster. Ethylmethane sulfonate (EMS) and dimethyl sulfate are also favorite mutagens among geneticists.


Example of a frameshift

met phe gln gln phe

ATG TTT CAG CAA TTT

met val ser ala ile

ATG GTT TCA GCA ATT T


Diseases of DNA repair

• Ataxia-Telangiectasia • Bloom Syndrome • Cockayne Syndrome • Fanconi Anemia • Xeroderma Pigmentosum• Hereditary non-polyposis colorectal

cancer

http://www.dnalc.org/resources/3d/index.html


Review

• Nucleotides and Nucleic Acids

- Structure and functions of nucleotides

- Important discoveries which helped solving DNA structure

- Major features of the DNA double helix


Review

• Structure of DNA

- ABZs of DNA structure- Primary, - Secondary and - Tertiary structure of DNA


Review

• DNA replication

- Major features of DNA replication

- Enzymology of DNA replication

- Eukaryotic DNA replication


Review

• DNA recombination and repair

- Enzymology of DNA recombination

- Major types of DNA recombination and repair

- Molecular basis of mutations

Garrett and Grisham, Biochemistry, Third Edition DNA Metabolism: Replication, Recombination, and...

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