Study Guide 2

Understand the 5 types of repair systems, and the general mechanisms of repair for each. [Focus more on enzyme functions (endo vs. exonuclease, ligase, helicase, polymerase) rather than on names of the proteins (AP1, FEN1, XRCC1, UvrABCD)].

1. Direct repair - direct reversal of damage (Example: Photoreactivation by photolyases of pyrimidine dimers 2. Removal and Replacement - base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (Primarily used to correct replication errors, preferentially correct the daughter strand) 3. Recombination Repair - retrieve undamaged copy that is then used to replace a damaged duplex sequence 4. Nonhomologous end joining (NHEJ) - to rejoin broken double-stranded ends 5. Resynthesis or bypassing of damaged DNA by DNA polymerases - Enzyme I (major repair enzyme), Enzyme II (replication restart), Enzyme III (replicase), Enzyme IV (translesion replication), Enzyme V (translesion replication) Error prone polymerases allow replication to continue without repair.

Be able to define what a mutation is, how mutations occur, the types of mutations.

An alteration in the DNA sequence that makes up a gene. May also involve the insertion or deletion (indels) of short sequences of DNA. Point Mutation - a change in a single base pair. can be caused by a chemical modification that changes on base into a different base or errors that occur during replication.

Know what DNA recombination is and why it is important

Bring together genetic material from multiple sources creating sequences that would not otherwise be found in the genome.

In terms of polymerases, know what each of these terms means, or that they are useful for: processivity, 3'-5' exonuclease, 5'-3' exonuclease.

DNA polymerase - An enzyme that synthesizes a daughter strand(s) of DNA (under direction from a DNA template). Any particular enzyme may be involved in repair or replication (or both) Processivity - The ability of an enzyme to preform multiple catalytic cycles with a single template instead of dissociating after each cycle

Know what DNA replication is, why it is necessary and when (in the cell cycle) it occurs

DNA replication is the process of DNA making a copy of itself. It is necessary so that genetic material gets transferred from the parent chromosome to daughter chromosomes. In Interphase (G1 + S + G2), replication occurs at the S phase.

Know that modified bases are a major cause for spontaneous mutations (hotspots), and why (using the example of deamination of cytosine vs. 5-methylytosine).

Deamination involves the removal of an amino group. The most common is the modified base 5-methylcytosine, which is spontaneously deaminated to thymine. Deamination of cytosine produces uracil, whereas deamination of 5-methycytosine produces thymine.

Know the types of DNA Polymerase errors

Error-prone Polymerase - A DNA polymerase that incorporates noncomplementary bases into the daughter strand Lesion Bypass - Replication by an error-prone DNA polymerase on a template that contains a damaged base. The polymerase can incorporate a noncomplementary base into the daughter strand. Proofreading - A mechanism for correcting errors in DNA synthesis that involves scrutiny of individual units after they have been added to the chain.

Know how bacteria and eukaryotes differ in leading and lagging strand synthesis and priming.

Eukaryotes - Continuous synthesis on the leading strand (5'-3'), Discontinuous on the lagging strand. Synthesized by making short fragments that are subsequently joined together. Leading strand requires only 1 priming event, lagging strand requires its own start and each primer sequence is ~10 bases long. Bacteria - The core on the leading strand is processive because its clamp keeps it on the DNA. Disassociation and re-association required on the lagging strand

Know the role of endonucleases, exonucleases, helicases, glycosylases, lyases and polymerases in repair of DNA damage.

Glycosylases hydrolyze the bond between base and deoxyribose Error prone polymerases allow replication to continue without repair Lyases open the sugar ring at the site of a damaged base APE1 endonuclease recruits DNA pol B to replace the single nucleotide. Cleaves chain on 5' end Exonucleases digest the nonmethylated strand until it passes the site where MutS is bound, removing the incorrect base.

Know the 2 types of recombination that we discussed in detail. Be able to name the third type of recombination

Homologous Recombination - involves reciprocal exchange of DNA sequences, between two chromosomes that carry the same genetic loci Site-specific recombination - occurs between two specific sequences Somatic Recombination - Occurs in non germ cells

Know what homologous recombination is and be able to describe important steps/aspects of homologous recombination, including how recombination is achieved, and what steps are necessary.

Homologous Recombination involving a reciprocal exchchange of sequences of DNA. Can occur at any point along the lengths of two homologous DNA. Involves two duplexes of DNA. Synapsis (pairing) and crossing over occur. Complementarity between single strands is required. Recombination frequency depends on chromosome structure. HR occurs during prophase I. Each chromosome has completed replication and consists of two sister chromatids, homologous chromosomes synapse (pair) to form bivalents (contain all four chromatids). Recombination forms a joing molecules between non-sister chromatids. Joint molecules resolve to form intact chromatids that have new genetic material. Double Strand breaks initiate recombination. In one DNA duplex is initiated by an endonuclease or occur spontaneously as a result of DNA damage. 5' end resection exonucleases generate 3' single stranded termini. one of the 3' single stranded ends invades a homologous region in the other duplex catalyzed by RecA -type proteins. A single strand from one duplex displaces its counterpart in the other duplex, it creates a D-loop. New synthesis replaces any material that has been degraded. This generates a recombinant joing molecule in which the two DNA duplexes are connected by heteroduplex DNA and two Holliday Junction. The joints are resolved by cutting.

Know the 3 main DNA-dependent DNA polymerases in bacteria and their polymerization and exonuclease activity

I - Major repair enzyme (polA) II - Replication restart (polB) III - Replicase (polC) Polymerization 5'-3' = I,II,III Exonuclease 3'-5' = I, II, III Exonuclease 3'-5' = I 5'-3' exonuclease activity of DNA pol I is not ideal for many applications. Proteolytic cleavage removes the small fragment (5'-3' exonuclease) and retains a large fragment (Klenow fragment) with DNA polymerase and 3'-5' exonuclease activity

Be able to briefly describe termination of replication in E.coli.

In E. Coli, the two replication forks usually meet halfway around the circle.

Know and be able to describe the steps of DNA replication: Initiation, Elongation and Termination.

Initiation - begins at the replication origin, the parental strands must be separated and transiently stabalized in the single stranded state - creates a "replication bubble" Elongation - Undertaken by the replisome: a large complex of proteins, as the replisome moves along DNA the parental strands unwind and daughter cells are synthesized Termination - Following termination, the duplicate chromosomes must be separated from one another

Understand and be able to describe the following terms and processes: origin of replication, priming, leading and lagging strand, unidirectional vs. bidirectional replication, replicon, replication bubble, semiconservative replication, Okazaki fragments, semi-discontinuous replication.

Origin of Replication - A sequence of DNA at which replication is initiated Priming - Before DNA polymerases can work they need a stretch of nucleic acids to start (often of RNA) Leading -The strand of DNA is synthesized continuously in the 5' to 3' direction Lagging - The strand of DNA that must grow overall in the 3' to 5' direction and is synthesized discontinuously in the form of short fragments (5'-3') that are later connected covalently Replicon - A unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication. Replication Bubble - A region in which DNA has been replicated within a longer, unreplicated region Semiconservative Replication - Replication by separation of strands of a parental duplex, with each strand then acting as a template for synthesis of a new complementary strand. Okazaki Fragments - Short stretches of 1000 to 2000 bases produced during discontinuous replication; they are later joined into a covalently intact strand Semi Discontinuous Replication - The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously Unidirectional replication: a single replication fork is created at an origin Bidirectional replication: when an origin creates two replication forks that move in opposite directions (usually the case)

Know which types of mutations are reversible and which are not

Point mutations and insertion can revert but deletions cannot revert.

Know how primers are removed and Okazaki fragments are linked/joined together

Primase synthesizes RNA, DNA polymerase III extends RNA primer into Okazaki fragment, next Okazaki fragment is synthesized, DNA polymerase I uses nick translation to replace RNA primer with DNA, ligase seals the nick. Each Okazaki fragment starts with a primer and stops before the next fragment. DNA ligase makes the bond that connects the 3' end of one okazaki fragment to the 5' beginning of the next fragment.

Know what site-specific recombination is, how site-specific recombination is achieved between bacteriophage and bacteria, and the names of sequences involved in integration and excision.

Recombination that occurs between two specific sequences, as in phage integration/excision or resolultion of cointegrated structure during transposition. Sitespecific recombination catalyzed by recombinases and enzymes involved in phage integration are known as integrases. Phage lambda integrates into the bacterial chromosome by recombination between sites on the phage and on the e. coli chromosome. Bacterial att sites: attB consisting of BOB' Phage att sites: attP consisting of POP' Recombination occurs within core sequence "O" common to attB and attP, attL and attR are products of recombination

Understand the basic mechanism of replication including the functions and importance of the following enzymes Topoisomerase, Helicase, Single Stranded Binding Protein, Primase.

Replication fork is initiated at the origin by helicase and then moves along DNA, A replicated region appears as a replication bubble flanked by nonreplicated DNA, SSB then binds to strands. DNA polymerase synthesizes new strand Topoisomerase - An enzyme that changes the number of times the two strands in a closed DNA molecule cross each other. Relieves positive super coiling ahead of replication fork. It does this by cutting the DNA, passing DNA through the break, and resealing the DNA. Helicase - An enzyme that uses energy provided by ATP hydrolysis to separate the strands of nucleic acid duplex Single-strand Binding Protein (SSB) The protein that attaches to single-stranded DNA, therby preventing the DNA from forming a duplex Primase - A type of RNA polymerase that synthesizes short segments of RNA that will be used as a primers for DNA replication

Know how replication is similar or different in bacteria vs. in eukaryotic chromosomes.

Similarities - Has DNA polymerase synthesizes new strands, leading and lagging strand synthesis. Differences - Each monomeric unit has a catalytic core, a dimerization subunit, and a processivity component. One Catalytic core is associated with each template strand. Eukarytoes use DNA polymerase α,delta, ε while bacteria use DNA polymerase III

Know the direction of synthesis in terms of 5' and 3' orientation (newly synthesized strand and of template strand)

Template strand = 5'-> 3' Leading Strand = 5'-> 3' Lagging Strand = 3'-> 5'

Know the role and types of Topoisomerases in recombination

Topoisomerase alter/resolve supercoiling by breaking bonds in DNA, changing the conformation of the double helix in space and remaking the bonds Type II enzymes act by making double strand breaks. Can pass a duplex DNA through a double-strand break in another duplex.

Know the general difference between the two types of point mutations.

Transition - a change of a pyrimidine (C,T) to another pyrimidine or a purine (A,G) to anotehr purine. Transversion - a change of a pyrimidine to a purine or vice versa

Know the 3 main DNA-dependent DNA polymerases in eukaryotes, and their role in leading and lagging strand synthesis.

α - Nuclear Replication (350kD tetramer) Delta - Lagging Strand (250 kD tetramer) ε - Leading Strand (350 kD tetramer) "Priming"/Initiating Replication