Biol 304 Exam 2 Study Guide WORD PDF

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BIOL 304 Exam 2 Study Guide (Sections 2.1 DNA Replication, 2.2 Telomere Replication, 2.3 DNA Recombination, 2.4 DNA Repair, & 2.5 Transposons & Retroviruses) -DNA Replication: The process by which a double-stranded DNA molecule is copied to produce 2 identical DNA molecules in order for a cell to properly divide -Initiation: Involves the recognition of an origin by a complex of proteins, strand separation of parental DNA into single strand templates through replication bubble formation, and proper establishment of a replication fork by several replication-associated proteins -Elongation: Established by a complex of proteins that function after replication fork formation (only associated with the particular structure that DNA takes at this site) and this complex comes along template strands of DNA and progressively unwinds the DNA, synthesizing new complementary daughter DNA strands 1. A precursor nucleotide enters the position at the end of the growing chain 2. A bond is formed and the enzyme moves forward 1 base 3. If a mistake has been made, the DNA is structurally warped by the incorporation of the incorrect base that will cause the polymerase to pause or slow down 4. The change in structure allows for the enzyme to potentially back up and remove the incorrect base -Termination: At the end of the replicon (unit of genetic replication), joining and/or termination reactions take place in order for duplicate chromosomes to be separated from one another through

manipulation of higher order DNA structure -Replisome: The complex of proteins that associates only at the replication fork of DNA and function to progressively unwind and synthesize new daughter DNA strands from the 2 template single strands of DNA -Topoisomerase: A set of enzymes that allows for the alteration to the supercoiled structure of a chromosome in order for replication to begin by making a cut, or nick, in either 1 or both DNA strand(s) -Topoisomerase I: A class of topoisomerases that nicks 1 strand of a DNA double helix

(these topoisomerases do not require ATP for hydrolysis) -Topoisomerase II: A class of topoisomerases that nick both strands of 1 DNA double helix (utilize ATP hydrolysis) -DNA Gyrase is a Topoisomerase II class enzyme that makes dsDNA nicks

-Replicon: A DNA molecule or RNA molecule, or region of DNA or RNA, which replicates from an origin of replication and may have a termination site -Eukaryotic or iSi t e:( Or i gi nofr epl i cat i onsi t e)Speci ficDNAsequences wher er epl i cat i onbegi ns Pr okar yot es,phages,pl asmi ds,Fepi somes ,and mi t oc hondr i al / c hl or opl as tDNAhav e1or isi t e Repl i c at i onatt heor i s i t ebegi nswi t ht hef ol l owi ngs t eps : 1.DnaApr ot ei nsbi ndt oor i C 2.Hel i cas eunwi ndsdoubl ehel i x 3.St r andsar esepar at ed 4.7ot herr epl i s omepr ot ei nspar t i c i pat ei nr epl i c at i on i ni t i at i on DnaA,DnaB,DnaC,HU,Gyr ase,SSB,&DnaG

chromosomes have 10’s of thousands of ore sites called Autonomous Replicating Sequences (ARS) -(Think of eukaryotic chromosomes as a cluster of replicons that undergo a coordinated start during “S” phase and complete replication in ~6 hours)

-Autonomous Replicating Sequence (ARS): The specific DNA sequence in eukaryotes where DNA replication begins (specific term for a eukaryotic ori site) -ARS elements are AT-rich with 4 domains: B3, B2, B1, and A -Origin recognition complex (ORC): a multi-subunit DNA-binding protein complex that recognizes ARS -A & B1: An 11-bp origin recognition sequence, contacted by the ORC throughout most of the cell cycle -B2 & B3: Domains that are only bound by the ORC when it is time to replicate DNA -The entire complex engages B1 and B2 and recruits other replisome components for DNA replication to proceed -Licensing factors: Proteins that are necessary for coordination of the cell cycle) called “licensing” factors” because, initially, biologists only knew they were needed for some part of DNA replication and thus termed them analogous to fishing: “you need a license to fish”) -Cdc6 Protein: (Cell division cycle 6 protein) A licensing factor protein (associated with Cdt1 and the ORC) that helps to coordinate the building of the ORC around a eukaryotic ARS for DNA replication to begin, specifically building the ORC at the ARS in the nucleus -Cdc6 is classified as an ATPase enzyme -Cdt1 Protein: (Chromatin licensing and DNA replication factor 1) A licensing factor protein present normally in the cytoplasm, that binds a helices and later associates with Cdc6 and the ORC in order for formation of a replisome -> initiation of DNA replication can occur -A Cdt1/helicase complex must enter the nucleus

to bind Cdc6/ORC

-Cdc6 and Cdt1 licensing factors are only synthesized (genes transcribed/translated) during late mitosis and the start of G1 phase because at this time in a eukaryotic cell there is a low [proteases], meaning less possibility of their degradation -Cdc6 and Cdt1 disengage from the ORC after replication starts -The process of degradation and re-synthesis of licensing factors throughout the cell cycle favors replication during the “S” phase -Semi-conservative Replication: DNA replication in which the 2 strands of the parental duplex are separated,

and each serves as a template for synthesis of a new, distinct daughter strand -Each parental strand results in a pair with a daughter strand: A final product of 2 complete chromosomes contains 1 parental strand and 1 newly synthesized strand

-S Phase: The synthesis phase of eukaryotic mitotic Interphase when DNA synthesis (replication) occurs, and replicon firing must be coordinated -DNA Polymerase: An enzyme that can synthesize a new DNA strand on a template strand (more specifically known as a DNA-dependent DNA polymerase) -Synthesis: Antiparallel replication activity from 5’ to 3’ from a template that is read and processed 3’ to 5’, adding nucleotides one at a time to a free 3’ -OH end -Energy is provided by the hydrolysis of PO4 bonds from the nucleotide before formation of a phosphodiester bond (forms the new replicate strand backbone) -Pyrophosphate (PPi): Two ester-linked PO4 groups that are released in the synthetic steps of nucleic acid elongation, as well as in protein elongation

-Prokaryotic Genome: Prokaryotes complete cell division through binary fission, as opposed to eukaryotic mitosis -Division is preceded by chromosome replication from a single ori site -i.e. E. coli cells can divide every 20 minutes (under optimal growth conditions) - ~5000 genes = 4.6 x 106 bp, for a DNA synthesis rate of 1000 bp/sec

-Parent and daughter DNA molecules are identical (except for mutations) -Prokaryotes are more concerned with speed of replication over accuracy (proofreading) -Mutations occur at a rate of ~1 mutation/chromosome/generation -Short generation times result in greater mutations: ~107 - 108 mutations/12 hours -Prokaryotic DNA is structured as a large, circular dsDNA molecule

VS

-Although some bacterial species have 2 chromosomes

-1 ori site per chromosome (typically 1 ori site per cell) -Plasmids, circular phages, mtDNA, ctDNA, are also circular chromosomes that have a single ori site

-Theta structure: The description of a circular prokaryotic chromosome undergoing replication, where the newly synthesized DNA molecule is jutting out from the parental chromosome at both replication forks, resembling the Greek letter Θ -Catenane: The term used to describe the nature of a parent prokaryotic circular chromosome where the 2 circular DNA molecules are interlocked (like the links of a chain) -Occurs when 2 replication forks move in opposite directions and stop at unique terminator sites, forming interlocking circular DNA molecules -E. coli use topoisomerase IV to make a dsDNA cut in 1 of the chromosomes to separate interlocked DNA molecules

-E. coli has 5 major DNA polymerase enzymes/holoenzymes: -DNA Polymerase III: (encoded in E. coli by dnaE locus, polC gene) A well-characterized E. coli holoenzyme that is the replication polymerase responsible for its de novo synthesis of new strands of DNA and elongation of daughter DNA strands -Conceptualized with the analogy of a “human hand”, with 5 domains: -“Palm” domain: The catalytic active site of DNA pol III with important conserved motifs -“Fingers” domain: Involved in positioning the template correctly at the active site (DNA is held in position by contacts made principally with the phosphodiester backbone) -“Thumb” domain: Binds the DNA as it exits the enzyme and ratchets the growing strand forward for continual synthesis, also important in processivity (facilitates proofreading) -ε subunit: (encoded in E. coli by the dnaQ gene) The 3’ to 5’ proofreading exonuclease subunit, distant from the actual catalytic site of the Palm domain, and the holoenzyme alternates from polymerizing and editing modes continuously through rotation after the substrate binds -When a mismatched base pair occupies the ε subunit catalytic site, the Fingers domain cannot rotate toward the Palm to bind the incoming nucleotide, and the exonuclease is able to act (rotates to exonuclease) -N-terminal domain: A spacer between catalytic synthesis and exonuclease domains

-Transient attractions between amino acids of the protein complex and the phosphodiester DNA backbone facilitate enzymatic activity

-DNA Polymerase I: An E. coli DNA polymerase encoded by pol A that is involved in the removal and replacement of primers, repair of damaged DNA, (and in a subsidiary role, semi-conservative replication also) -DNA polymerase I is a single nucleotide peptide of 103 kD, which can be cleaved through proteolytic treatment into 2 parts, which both coordinately work to provide DNA pol I with the unique ability to start replication in vitro at a random nick in DNA (NO OTHER DNA POLYMERASE HAS THIS ABILITY) -Klenow fragment: The larger cleavage product (68 kD) that is used in synthetic reactions in vitro and shown to contain the polymerase and proofreading 3’ to 5’ exonuclease activities -Small fragment: The smaller (35 kD) fragment of DNA pol I that possesses 5’ to 3’ exonucleolytic activity, excising small groups of nucleotides of up to ~10 bases at a time -Nick translation: The ability of DNA pol I to recognize a break in a phosphodiester bond in DNA and extend the available 3’ -OH end, displacing and degrading the existing homologous strand in the duplex by 5’ to 3’ exonucleolytic activity (mainly used in lagging strand DNA replication to fill in short segments where DNA is single-stranded) -[DNA polymerase I is the predominantly exhibited enzyme when E. coli extracts are assayed for their ability to synthesize DNA with activity so great that is impossible to detect the actual polymerases responsible for DNA replication (possibly why it was the 1st characterized DNA polymerase, termed “I”); mutant extracts from pol must be prepared to develop an in vitro system for analysis in lab]

-DNA Polymerase II: An E. coli DNA polymerase encoded by the polB gene that is required to restart a replication fork when its progress is stalled/blocked by damage in DNA -DNA Polymerase IV and V: (encoded by dinB and umuD’2C, respectively) E. coli-derived DNA polymerases that are involved in DNA repair and allowing replication to bypass certain types of DNA damage (known as error-prone polymerases involved in translesion replication) -Eukaryotic polymerases: Several polymerases that are involved in nuclear replication, priming, repairing damaged nuclear DNA, or in translesion replication of damaged DNA when repair is impossible -DNA polymerase γ: A mitochondrial DNA polymerase that carries out general replication of the organellar genome (chloroplasts have their own replication system)

-5 E. coli Polymerases Summarized: DNAPol1:Repai r ,j oi ni ngofOkazaki f r agment s( si ngl esubuni tenzyme) DNAPol2:Rest ar t i ngofst al l edr epl i cat i onf or ks( si ngl esubuni tenzyme) DNAPol3:Maj orr epl i case,par toft heor i Cr epl i s ome( mul t i s ubuni thol oenzyme) DNAPol4&5:Tr ansl es i onpol y mer ases , Repai r( si ngl esubuni tenzymes) -Holoenzymes: (see left) Large, multi-protein assemblies that have both DNA-synthesizing subunits and also DNArepair subunits, as well as serving several other functions concerned with fidelity, or replication reliability (holoenzymes are advantageous because of the availability of a specialized subunit on hand for error correction, reducing overall rates of replication errors)

-Eukaryotic DNA Polymerases: Several DNA polymerases utilized by eukaryotes and each polymerase has different functions, and is employed at different times; involved in replication, recombination, and DNA repair (and > 1 polymerase is active at any given time in a cell); 3 polymerases carry out most of nuclear DNA replication: -Alpha (α): Eukaryotic DNA Pol that initiates nuclear DNA synthesis/DNA repair, with primase activity: synthesizing an RNA primer followed by 30-40 DNA nucleotides (NO exonuclease activity) -Delta (δ): Eukaryotic DNA Pol that replicates on the lagging strand and has 3’ to 5’ exonuclease activity, also involved in DNA repair and translesion DNA synthesis -Epsilon (ε): Eukaryotic DNA Pol that replicates the leading strand of DNA and has 3’ to 5’ exonuclease activity -Physical design of DNA polymerases: Presence of 2 simultaneously synthesizing catalytic enzyme cores is the reason for the leading and lagging strand dichotomy

-DNA polymerase(s) are dimers: One monomer is responsible for leading strand synthesis and the other monomer is responsible for lagging strand synthesis -Both DNA strands are synthesized concurrently by looping the lagging strand to invert the physical, but not biological direction of synthesis -Leading Strand: Sometimes called the forward strand, this is the strand of DNA that is replicated continuously in the 5’ to 3’ direction, as the parental duplex is unwound (CONTINUOUS REPLICATION) -Lagging Strand: The template strand in which a stretch of DNA is exposed, and a segment is synthesized in the reverse direction (relative to fork movement); a series of these fragments are synthesized, each 5’ to 3’, and then joined together to create an intact daughter strand (DISCONTINUOUS REPLICATION) -Okazaki fragments: The short

fragments of ~1000 to 2000 bases in length synthesized on the lagging template strand, found both in prokaryotes and eukaryotes, that are later covalently linked together to form a single complete daughter strand of DNA -Semi-Discontinuous Replication: The further-refined term for the replication of DNA, taking into account continuous replication of the leading strand and the discontinuous replication of the fragments of the lagging strand -Important Replication Proteins/Structures: Involved in both eukaryotic and prokaryotic DNA replication -Helicase: (blue triangle) An enzyme that separates (or melts) the strands of DNA, usually using the hydrolysis of ATP to provide the necessary energy -There are 12 different E. coli helicase enzymes that are generally multimeric, but most commonly seen in the form of a hexamer

-1 conformation of helicase tends to bind to duplex DNA and another conformation binds to ssDNA, and alternation between formations drives the motor that melts the duplex apart -Helicase usually initiates unwinding of DNA at a single-stranded region adjacent to a duplex (helicase cannot unwind a segment of duplex DNA) and can only continue to unwind a sequence of DNA that has already separated -Helicase unwinds with either a 5’ to 3’ (5’—3’ helicase) or 3’ to 5’ (3’—5’ helicase) polarity -Single-Strand Binding (SSB) Protein: (purple oval) A protein that binds to the ssDNA after unwinding a helicase, protecting it and preventing it from reforming the duplex state -E. coli SSB is a tetramer of 74 kD, while the eukaryotic SSB (termed RPA) is a trimer -SSB binds ssDNA cooperatively, where binding of 1 protein molecule makes it much easier for following molecules to bind -Under normal circumstances in vivo, unwinding, coating, and replication reactions all proceed continuously in tandem -Synthesis of a new strand can only start from a preexisting 3’ —OH end -DNA Gyrase: (green disk) An endonuclease protein that moves ahead of the replication fork, making and resealing breaks in the double-helical DNA to release the torque that builds up as a result of unwinding at the replication fork (formation of a replication bubble leads to increased strain on the chromatin on either side) (DNA Gyrase) -E. coli gyrase proteins are encoded by the genes gyrA and gyrB -E. coli gyrase is a type of topoisomerase II -DNA Ligase: (light blue oval) An enzyme that covalently links together fragmented DNA by making a bond using a complex involving AMP -The AMP of the enzyme complex becomes attached to the 5’ phosphate of the nick and then a phosphodiester bond is formed with the 3’ —OH terminus of the nick, thus releasing the enzyme and AMP -Ligases are present in both prokaryotes and eukaryotes -E. coli and ΦT4 ligases share the property of sealing nicks that have 3’ —OH and 5’ —phosphate termini: 1. The AMP of the enzyme complex becomes attached to the 5’ phosphate of the nick

2. A phosphodiester bond is formed with the 3’ —OH terminus of the nick, releasing the enzyme and the AMP -Primer: The free 3’ —OH end of newly synthesized strand (required by both lagging and leading strands) that allows for the elongation of a new daughter strand by way of DNA polymerase, which is created by multiple mechanisms (a common feature of all DNA polymerases is that they cannot initiate synthesis of a chain of DNA de novo, but can only elongate a chain): -RNA primer: A sequence of RNA that is synthesized on the template, creating a free 3’ —OH end, commonly used in replication of cellular DNA and some viruses -Preformed RNA (often a tRNA): An already synthesized RNA molecule that pairs with a template strand, providing its 3’ —OH end to be used to prime DNA synthesis, utilized by retroviruses to prime reverse transcription of RNA -Primer terminus (DNA-derived): A sequence of DNA -Methylation regulates initiation of replication in that is generated from within the template duplex, the oriC system, and only fully methylated most commonly created by introduction of a nick in sequences in both strands can initiate the DNA, and is used to initiate rolling cycle replication replication (the preexisting strand is displaced by new -Dam methylase: A chemical modification synthesis) enzyme that adds methyl groups to -Protein primer: A protein molecule primes the nucleotides, specifically Adenine, in order to replication reaction directly by presenting a help trigger replication in E. coli nucleotide to the DNA polymerase, utilized -Methylation in the figure on the right is shown by certain viruses by the green “m”

-The leading strand of DNA only requires 1 priming event, at the origin of replication -The lagging strand of DNA requires a series of initiation events because each Okazaki fragment requires its own start site (each with a priming sequence of ~10 bases long)

-Primase: An enzyme that is required to catalyze the actual priming reaction for DNA synthesis -The E. coli primase DnaG (encoded by the dnaG gene), is a single polypeptide RNA polymerase enzyme of 60kD that associates transiently with the replication complex and synthesizes short (11 ± 1 base) primers, providing a free 3’ —OH group for daughter strand elongation -E. coli primers start with the sequence pppAG positioned opposite the sequence 3’—GATC— 5’ in the template and have 2 types of priming reactions: 1. oriC system: (bacterial origin) Involves the association of the DnaG primase with the protein complex at the replication fork -E. coli oriC contains 11 copies of a 5’—GATC—3’ sequence: all of which are targets for methylation by Dam methylase -N6 position of A in the 5’—GATC—3’ is methylated

-Difference in the figure on the left is only the amount of methylation at the E. coli oriC sites (resulting in regulation of replication initiation) -Methylation is depicted by the pink dots on the DNA doubl...