BIOL200 dna shit

how do nucleotides get added to a growing dna strand?

dNTP phosphate group reacts with 3’ OH group in the growing chain

what is the name of the molecular machine that carries out DNA replication?

replisome

what enzyme is responsible for splitting the DNA strand in eukaryotes? what are its subunits? where does it bind?

• CMG helicase

• Cdc45 and GINS complex subunits

• binds to the leading strand

what is the function of replication protein A (RPA)?

• binds to single strand to keep it in the optimal position for DNA polymerase

what is positive supercoiling? how is it solved?

• opening of the DNA helix introduces twists ahead of the replication fork

• this inhibits replication

• topoiserases cut pieces of DNA, allow the strand to pass through, and restitch

what does DNA polymerase require to function?

• binding to a primer

• dNTPs

• 5’-3’ direction

what are primers usually made of? what enzyme creates primers?

• RNA

• primase

what enzyme extends RNA primers?

DNA polymerase $\alpha$ forms a complex with the primer and extends it with DNA

what enzyme extends the leading DNA strand?

DNA polymerase $\epsilon$

which enzyme replaces the RNA primer with DNA?

DNA polymerase $\delta$

compare the functions of the different types of DNA polymerase

• DNA pol $\alpha$ extends RNA primers with DNA

• DNA pol $\epsilon$ extends the leading strand

• DNA pol $\delta$ replaces primers with DNA

what is the function of PCNA?

ring shaped protein which prevents DNA pol from detaching from the template

what is the function of RFC?

opens PCNA and clamps it to the template at the location of the primers

what enzymes remove RNA from okazaki fragments? which enzyme replaces it with DNA? which enzyme connects the fragments?

• ribonuclease H and FEN-1 remove RNA

• DNA pol $\delta$ replaces with DNA

• ligase binds fragments

where are does DNA replication begin? what is special about these sites?

• origins of replication

• AT rich

what protein marks the origin of replication? what does it do and during what phase of replication?

• origin recognition complex (ORC)

• loads helicase during G1 phase in inactive form

• activates during S phase

what are characteristics of mutations?

• permanent

• transmissible

how can mutations occur?

• spontaneously via transposable elements or replication errors

• by mutagens → i.e. radiation or chemicals

which DNA polymerases have proofreading?

DNA pol $\epsilon$ and $\delta$

what is the process of a mismatch repair?

• mismatch creates a deformity

• MSH2 and MSH6 bind on the daughter stand on either side of the mismatch

• MLH1 endonuclease cuts around it

• pol $\delta$ and ligase fills the gap

why is PCNA important in a mismatch repair?

• signals for repair near the replication site

• directs repairs to newly synthesized strands

• helps with MSH and MLH binding

what is an example of a situation where a base excision repair might be needed?

• the deamination of cytosine can create a uracil unit

what is the process of a base excision repair?

• DNA glycolases hydrolyzes the bond between the base and the backbone

• APE1 cuts the backbone

• AP lyase associated with DNA pol $\beta$ removes backbone fragment

• DNA pol $\beta$ and DNA ligase fill the gap

what gives base excision repairs specificity?

11 types of DNA glycolases recognize different types of errors

what is an example of when a nucleotide excision repair might be used?

thymine dimers create bulky regions required repairs

what is the process of a nucleotide excision repair?

• 23B and XP-C bind to the repair site

• TFIIH, RPH and XP-G unwind the region

• XP-F and XP-G cuts either end

• pol $\delta$ and ligase fill the gap

what is the issue with errors on DNA? how is this solved?

• during replication DNA pol can’t continue after an unfixed error

• TLS polymerases can bypass it, but often make the wrong base pairing, introducing a mutation

what problem do chromosomes solve?

unstructured DNA is very voluminous and the strands are hard to separate

what is the hierarchy of chromosomal folding?

• nucleosomes

• chromatin

• chromosomes

how are nucleosomes formed?

DNA (-) wrapped around a histone protein core (+)

what is the composition of histones?

octamer

• H2A, H2B, H3, H4

• 2 of each

what is the function of histone H1?

links nucleosomes → “beads on a string”

what is the difference between chromatin structure in metaphase vs interphase?

• metaphase → compacted

• interphase → decondensed

what are the different forms of chromatin?

• heterochromatin → condensed, repetitive and less genes

• euchromatin → decondensed, less repetitive, more genes/transcription

how are protein loops formed? what is special about them?

• SMC protein complexes act as a scaffold for loops to form

• loops are high in transcription

what is a structural difference between the different types of histone proteins?

• H2A & H2B → both N and C terminals

• H3 & H4 → only N terminals

what do the post transcription modifications of histone tails do? what is an example?

• determines the condensation of chromatin → epigenetic regulation

• acetylation of lysine units decondenses chromatin → (+) amino group neutralized by the acetyl group

how do chromosome fibers form?

• condensing II creates a central scaffold

• condensing I further compacts into clusters

what are some important sites on a chromosome?

• origins of replication

• centromeres

• telomeres

what are centromeres made of?

• kinetochores and microtubules

• histone CENPA

what function do telomeres serve?

• protects from exonucleases

• solves shortening of lagging strand

how are telomeres formed?

• simple repeat sequences

• in germ and stem cells → telomerase

• somatic cells replication is limited

what is a transcription unit? what can appear before them?

• regions of DNA with coding and non coding sequences

• bound by an initiation and termination site

• control regions before the initiation site regulate transcription

what is alternative splicing? what are its products called?

• introns are removed from a gene by splicing

• splicing can occur at different locations to create different products

• different proteins produced by alternative splicing are called isoforms

how can multiple coding regions form the same protein?

recombination

what is it called when a gene copy loses its function?

pseudogenes

what is a homolog? what is an example? what are some types?

• when a gene copy takes on a new function

• $\alpha$ & $\beta$ -tubulin

• paralogs → closely related proteins within the same organism

• orthologs→ the same protein in different species

what are the types of SSRs?

• minisatellite DNA → longer repeat units → form centromeres and telomeres

• microsatellite DNA → shorter repeat units → found in transcription units

how does DNA fingerprinting work?

SSR locations vary highly between individuals

what are the major classes of transposable elements?

• transposons

• retro-transposons

what are transposons?

regions of DNA cut and pasted to another region

• bound by inverted repeats

• transposase cuts and inserts region

• DNA pol and ligase seal it

how can transposons increase in copy number?

if the transposon moves from an replicated region to a non-replicated one

what are retro-transposons?

genes copied and inserted into a new site. more of a copy paste than a cut and paste

• DNA transcribed to RNA

• reverse transcriptase converts that RNA to a DNA copy which gets inserted into the genome

what are the two major structures of retrotransposons?

• LTR retrotransposons have long repeated sequences on either end

• non-LTR retrotransposons include LINEs and SINEs

what are LINEs and SINEs?

• non-LTR retrotransposons

• LINEs contain encoding for proteins needed for their movement (reverse transcriptase) (ORFs)

• SINEs do not encode their own transcriptase but use ORFs at other sites

how are LTR retrotransposons moved?

• tRNA acts as a primer at the LTR

• reverse transcriptase creates DNA

• integrase integrates the DNA into a new region

describe the process of recombinant DNA technology

• a vector (i. e. plasmid) is combined with a DNA fragment → recombinant DNA

• replicated in a host cell

• isolated and purified

how does DNA replication differ in plasmids than chromosomes?

can happen at any time in the cell cycle

are are DNA fragments inserted into a vector?

restriction enzymes

• cut DNA at restriction sites

• palindromic sequence

• staggered cut creates sticky ends

in addition to the fragment DNA, what sequences are added to a plasmid?

• restriction marker → i.e. antibiotic resistance. only recombinant cells will survive

• polylinker → contains many restriction sites

what are the types of DNA libraries?

• genomic libraries

• cDNA libraries → mRNA sequences which only contain coding regions → cDNa via reverse transcriptase

what can sometimes be found in a protein expression vector?

• promoter → requires a specific molecule to begin transcription

• purification tag → helps purify the protein

what is the difference between transient and stable transfection?

• transient → plasmid introduced, not integrated into host genome. diluted as the cell divides

• stable → integrated into a chromosome. long lasting

what is unique about the prokaryotic chromosome?

• 1 circular chromosome with 1 OriR

• centromeres but no telomeres

• gene rich regions are grouped into operons which have a common promoter and create a single mRNA strand with multiple coding sequences

how is DNA compacted in prokaryotes?

no histone proteins

• some proteins to help compaction

• negative supercoiling does most of the compaction → DNA topoisomerase gyrase

• SMCs generate loops like in eukaryotes

why are mitochondria and chloroplasts thought to have originated from bacteria?

similar genomes

• one circular chromosome

• few introns

• similar products

what are the products of mitochondrial DNA? where do they end up?

• components for respiration

• products stay in the mitochondria → they have their own ribosomes

compare the sizes of mitochondrial and chloroplast DNA

• mitochondrial → very short (37 genes)

• chloroplasts → larger (100-200 genes)

what is endosymbiosis theory? what are likely ancestors of each the endosymbiote and endosymbiont?

• host ancestor → likely archaea → lokiarchaea in hydrothermal vents

• mitochondrial ancestor → rickettsia

• chloroplast ancestor → cyanobacteria

• host engulfed endosymbiont and a mutualistic relationship emerged

what proves that genes have moved from the mitochondria/chloroplasts to the nucleus over the course of evolution?

some mitochondrial/chloroplast proteins are coded for in the nucleus

describe cytoplasmic inheritance

• only the egg contributes a mitochondria during fertilization

• mitochondrial DNA is inherited maternally

which enzymes are coded for within transposons? what about retrotransposons?

• transposons → transposase

• LTR retrotransposons → integrase and reverse transcription (ORFs). SINEs don’t have ORFs

what sequences are in non-LTR retrotransposons

• no LTRs, UTRs with AT rich regions

• poly A tail like mRNA.

• LINEs have ORFs, SINEs do not

how do SINEs depend on LINEs

• SINEs do not code their own integrase or reverse transcriptase

• they use the ORFs in LINEs instead

what components are required for PCR?

• DNA template

• Taq polymerase

• primers

• dNTPs

what are the stages of PCR?

• denaturation → heat separates DNA strands

• annealing → temperature decrease allows primers to bind

• extension → taq polymerase extends the primers at a higher temperature

what does DNA’s melting temp (Tm) correspond to? what does it depend on?

• temperature where ½ of the DNA is single stranded

• depends on the number of H bonds

what are the primers in PCR made of?

DNA

what is taq polymerase? what is special about it?

• heat resistant DNA polymerase from Thermus aquaticus

• no proofreading activity

how do you determine the number of copies made by pcr?

N = N0 X 2^n

what often follows PCR?

gel electrophoresis

how can restriction sites be added to PCR products?

restriction sites can be added to the 5’ end of each primer

what are the main types of DNA sequencing? how much DNA does each require?

• sanger sequencing → requires lots of DNA

• next generation sequencing (NGS) / illumina sequencing → less DNA

• nanopore sequencing → sequencing of long molecules

what is the process of sanger sequencing?

PCR is run with a low concentration of ddNTPS

• introduction of a ddNTP ends replication

• creates fragments of different length, fluorescently tagged

• electrophoresis separates them by length

what is the process of illumina sequencing?

• linkers are ligated to each end of DNA

• linkers bind to primers, bound to a solid surface

• PCR performed to create lots of copies fixed in one place

• primer is added to the copied strand and fluorescently tagged dNTPs bind one by one

describe the process of nanopore sequencing

• long molecule passed through a tiny pore

• changes in electrical current correspond to monomers

• detects modifications

what is special about ddNTPs?

they lack a 3’ hydroxyl group → prevents the binding of the next nucleotide