Exam 4 DNA metabolism Flashcards
What is DNA metabolism?
Set of processes that achieve these tasks
- New DNA copy synthesized before each cell division
- Checking for errors that arise during/after DNA synthesis and repairing
- Segments of DNA rearranged within a chromosome or between two DNA molecules (recombination) which gives offspring a novel DNA
What are the 3 fundamental rules of replication?
- Replication is semiconservative (daughter strand made from parent strand, new DNA has parent and daughter strand)
- Begins at an origin and proceeds bidirectionally
- Synthesis of new DNA occurs in 5’ to 3’ direction and is semi-discontinuous
Meselson-Stahl experiment
- Proved Watson& Crick’s hypothesis of Semiconservative replication: new DNA has old (parent) strand and new (daughter) strand
- nitrogen used for synthesis of new dsDNA is equally divided between the two daughter genomes
Once replication begins, does it proceed in same or opposite directions?
- It is bidirectional (Has 2 replication forks)
- Both strands are replication simultaneously
- circular DNAs have extra loop
Can replication begin anywhere or does it begin in the same location?
Loops always initate/begin at same unique origin
- Inman’s experiment: denatured DNA at AT-rich regions -> “bubbles” that showed it starts at unique origin
What direction does synthesis proceed in?
- 5’ to 3’: always by addition of new nucleotides to the 3’ end (3’-OH)
- Leading strand: made continuously as replication fork moves
- Lagging strand: made discontinuously in short pieces (Okazaki) that’s later joined together
What are enzymes that degrade and create DNA segments?
- Nucleases: degrade nucleic acids (DNases: DNA, RNases: RNA)
- Exonucleases (from outside): cleave bonds that remove nucleotides from ends of DNA
- Endonucleases: cleaves within a DNA sequence
- Polymerases: build DNA strands (at least 5 DNA polymerases in E.Coli)
DNA elongation
- Template: parent strand
- Substrate: nucleoside triphosphates
- nucleophilic OH group at 3’ end of growing chain attacks a-phosphate of incoming trinucleotide (3’OH required and made more powerful by nearby Mg2+ ions)
- Pyrophasohate: good leaving group
DNA polymerase
- Needs a primer: shorter strand complementary to temple (contains 3’-OH to begin first reaction and made of RNA)
- Enzyme has pocket with 2 regions: insertion site (where incoming nucleotide binds), postinsertion site (newly made base pair resides when polymerase moves forward)
What is Processivity?
of nucleotides that DNA polymerase can add before dissociation (DNA poly. can add nucleotides or dissociate)
- Each polymerase has own processivity and polymerization rate
Geometry of Base pairing accounts for high Fidelity in E.Coli
DNA polymerase active site excludes base pairs with incorrect geometry but sometimes it’s still inserted - repair mechanisms fix this
What repair mechanisms fix errors during synthesis?
Errors during synthesis are corrected by 3’ -> 5’ exonuclease activity
- it proofread synthesis for mismatched base pair
- translocation of enzyme to next location is inhibited until the incorrect nucleotide is removed
5 DNA polymerases in E.Coli
- DNA Poly I: function is clean up (not ideal for replication bc slow replication fork and ow processivity (falls easily))
- DNA Poly III: replication polymerase
- DNA POly II, IV, V: DNA repair
DNA Polymerase I also has 5’ -> 3’ Exonuclease Activity (in addition to 3’ -> 5’ Exonuclease Activity)
- Moves ahead of enzyme and hydrolyzes things in its path
- Does nick translation (strand break moves along with enzyme)
(these happen in the Klenow fragment: domain that can be separated by Klenow fragment)
DNA Polymerase III
Replication Polymerase with 10 types of subunits (core domains linked by clamp-loader complex)
- Core domains interact with dimer of b-subunits that increase the processivity of the complex (by forming a sliding clamp that prevents dissocation)
What is required for E.Coli DNA Replication?
Replisome: made of over 20 enzymes and proteins
- Helicases (use ATP to unwind DNA), Topoisomerases (relieve stress caused by unwinding), DNA-binding proteins (stabilize separated strands), Primases (make RNA primers), DNA ligases (seal nicks between nucleotides on same strand)
Initiation of replication in E.Coli
- Beings at oriC site, contains highly conserved sequence elements made of 5 repeats of 9b-p sequence (R sites) that form binding site for DnaA (initiator protein)
- ## DNA unwinding element (DUE) A=T rich region
Regulation of Replication Initiation via Methylation
After replication: oriC methylated by Dam methylase (methylates N6 of A in GATC sequences)
- hemimethylated oriC seq. interact with plasma membrane using protein, SeqA
- SeqA dissociates and oriC sequences are released
- Dam fully methylates DNA allowing new DnaA to bind
How does elongation of the leading strand happen?
DnaG primase makes RNA primer, DnaG primase interacts with DnaB helicase but moves in the opposite direction to helicase, Dna Pol III adds nucleotides to the 3’ end of the strand (Pol III is linked to DnaB which is on the opposite DNA strand)
How does elongation of the lagging strand happen?
Primase makes RNA primer and DNA Pol III adds nucleotides to 3’ end of the new strand. This strand is elongated away from the replication fork
- One asymmetric DNA pol II dimmer complex synthesizes both strands
Synthesis of the Leading and Lagging Strand
- Core subunits of DNA POL II dissociate from one b-clamp and bind to new b-clamp
- RNA primer is removed by DNA pol I or Rnase H1 and DNA Pol I fills in the gap with DNA (where the RNA primer was)
- DNA ligase seals the backbone
What are the three stages of DNA replication?
Initiation, Elongation, Termination
Termination of DNA (Third step of DNA replication)
Replication forks meet at region with Ter sequences (Ter A-F made of 20 b.p)
- Ter sites are near but in opposite directions and creates a site where replication forks can’t leave
What does Ter sequence do?
- Causes replication fork to stop
- Also binding site for protein, Tus
Replication in eukaryotes vs. E.Coli
- More complex
- In yeast: has replicators or ARS (autonomously replicating sequences)
- Entire genome replicated 1X/cycle (regulation due to cyclin proteins and CDKs)
Initiation of DNA replication in eukaryotes
- Requires pre-replicative complex (pre-RCs)
- ORC (origin recognition complex) loads helicase onto the DNA (ORC is similar to bacterial DnaA)
- Helicase: hexamer of mini-chromosome maintenance proteins (MCM2-& acts like bacterial DnaB)
- Slower than in E.Coli
DNA polymerases in eukaryotic nuclear replication
DNA Pol a: polymerase/primase activity
- primase in one subunit, polymerization in other subunit
- no 3’-5’ proofreading
DNA Pol delta (lagging and leading strand): associated with PCNA
- highly processive, both have 3’-5’ proofreading
- DNA Pol epsilon: leading strand
How is replication terminated in eukaryotes?
Synthesis of telomeres are found at the linear ends of the nuclear chromosomes
- Telomerase (enzyme) uses RNA
DNA repair and mutations
- Majority of damage in genomic DNA is corrected by using the undamaged strand as the template
- Some base changes escalate repair and incorrect base serves as a temple -> this leads to the daughter DNA having a changed sequence in both strands (mutation)
- Accumulation of mutations is correlated with cancer (most carcinogens = mutagens)
What are DNA lesions?
Lesions = DNA damage
- If a lesion is unrepaired, it becomes a mutation
Mutations can be: substitutions (point mutations), deletions, additions, or silent mutations (no effect on gene function or affects nonessential DNA region)
What are the types of DNA damage that can happen?
- Mismatches: from occasional incorporation of incorrect nucleotides
- Abnormal bases: from spontaneous destination, chemical alkylation, exposure to free radicals
- Pyrimidine dimers: when DNA is exposed to UV light
- Backbone lesions: from exposure to ionizing radiation and free radicals
How do repair enzymes know which strand is the correct one? (mismatch repair replies on methylation)
In E.Coli: parent strand is methylated
- Dam methylase inserts CH3 at adenines in the GATC sequence. Then after some time, the daughter strand is also methylated
Why is the newly synthesized strand in methylated for a short period of time after synthesis?
- To repair any errors: any replication errors have to be in the unmethylated strand
- The methyl-directed mismatch repair system cleaves the unmethylated strand in the repair process (MutH cleaves the strand)
What is base excision repair?
- Uses specific DNA glycosylases (recognizes specific lesions and cleaves N-glycosyl bond between sugar and base -> creates AP site)
- Uracil glycosylase removes Uracil from DNA (impt. bc C deanimates to U, which is not in DNA)
- Other glycosylases make AP Sites at other locations
What is the process of Base-Excision Repair (BER)?
Entire nucleotide is removed, sometimes the region around the AP site is removed
- AP endonucleases cut DNA backbone around AP site and removes DNA
- DNA Pol I synthesizes new DNA
- DNA ligase seals the nick
Nucleotide Excision (NER)
Repairs large distortions in DNA
- Pathway: involves removal of DNA segment by exinucleases
- Lesions include: pyrimidine dimers & 6,4- photo products (from UV light), benzo[a]pyreneguanine (cig. smoke)
How does direct repair work?
- Photolyases: use light energy to repair pyrimidine dimers
- O6-methylguanine-DNA methyltransferase repiars methylated guanine
- AlkB demthylates 1-methylase nine and 3-methylcytosine
How does O6-methylguanine lead to mutation?
O6-methylguanine leads to mutation by causing a mispairing during DNA replication, where it preferentially pairs with thymine instead of its normal partner cytosine, resulting in a G:C to A:T transition mutation when the DNA is replicated further;
How to repair DNA if there’s no undamaged DNA strand to use as template?
- Unrepaired lesions can use replication fork to stall
Can repair using: - another chromosome as template (recombination)
- error-prone translesion synthesis (TLS)
What is error-prone TLS in bacteria?
- Part of the SOS response (happens when there’s extensive DNA damage)
- SOS proteins: UvrA, UvrB + UmuC and UmuD
- Cleaved UmuD and UmuC binds with RecA to make DNA Pol V, which can process past the damaged area
TLS polymerases in mammals
- Can recognize specific damage and have appropriate response (ex: DNA Pol eta when T-T dimer hats replication fork)
- Limited to short regions of DNA -> limits mutagenic potential
What is DNA recombination?
DNA segments can rearrange location within a chromosome or from one chromosome to another
- involved in the process of DNA repair, segregation of chromosomes (meiosis), enhancement of genetic diversity
- Recombination, Mutation = driving force of evolution
What does recombination of co-infecting viral genomes enhance?
- Virulance
- Provide resistance to antivirals
What are the three classes of DNA
recombination?
1) Homologous/General recombination
- exchange between two DNAs that share extended region of similar sequence
2) Site-specific recombination: exchange only at particular sequence
3) DNA transposition: “jumping genes” - short DNAs that move from one chromosome to another
What is Non-homologous end joining (NHEJ)?
Way to repair DSB (double strand break) but not ideal
- broken chromosome ends processed and ligated back together (no conservation of DNA sequence)
What is site-specific recombination?
- Limited to specific sequence: recombinase enzymes with Tyr or Ser
- Recombinase makes covalent bond with DNA, Cleaved strand joins new partners
- Reciprocal exchange
What are the potential outcomes of site-specific recombination?
- Inversion or deletion: if recombination sites are on the same DNA
- Intermolecular recombination: if recombination sites are on different DNAs
- Insertion: if the DNA is circular
What are transposable genetic elements (Transposons)?
Certain DNA segments can change relative position (to new region or chromosome) and they carry genes for transposases (but can contain extra genes
- Binding sites for transposases in bacteria: short (5-10b.p) sequences, and these sites become duplicated
Direct vs. Replicative Transposition
- direct transposition: “cut-and-paste” mechanism where a transposable element is excised from its original location and directly inserted into a new site, leaving only one copy of the element in the genome
- replicative transposition: involves copying the transposable element during the transposition process, resulting in a duplicate copy at the new insertion site and leaving the original copy intact
