Term
| Three postulated methods of DNA replication |
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Definition
Semi-conservative- 2DNA parental strands, one complete strand goes to one
new cell, the other goes to the other cell.
Conservative
Dispersive |
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Term
| DNA replication is ALWAYS synthesized in |
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Definition
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Term
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Definition
Template (parental ssDNA)
Primer
4 dNTPs -triphosphates
Buffer -needs Mg
DNA polymerase |
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Term
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Definition
| : The enzyme that adds one nucleotide at a time. It req the template and primer. Has to add nucleotide to a pre existing strand. Cannot start from scratch |
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Term
| Synthesis of DNA can occur ONLY in 5’ to 3’ direction. This means the template runs |
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Definition
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Term
| Template is 3’-5’ so synthesis is |
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Definition
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Term
| 3’ hydoroxyl on the last nucleotide (dGTP) the attaakts |
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Definition
| the P (alpha P of dTTP which has a positive charge on it from O attached to it) energy of making covalent bond comes from the 2nd and 3rd P come off as pyrophosphate* |
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Term
| 3’ OH on the 3’ of the growing daughter strand does a nucleophile attack on the |
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Definition
| incomming dNtp and pyrophosphate is cleaved off forming a new 5’ 3’ phosphodiester bond. |
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Term
| Base must pull off H (on 3' hydroxyl to make |
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Definition
| O more electronegetive to allow it to do Nucleophile attack. |
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Term
| Leading strand replication needs # of RNA primer |
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Definition
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Term
| RNA primer is added via -- and removed via -- |
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Definition
added by primase
removed by DNA polymerase 1 |
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Term
| DNA -- joins the Okazaki fragments by forming a -- |
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Definition
ligase
covalent 3'-5' phosphodiester bond |
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Term
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Definition
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Term
| What lengthens DNA replication? |
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Definition
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Term
| In bacteria, Okazaki fragments are ~--nucleotides long. In eukaryotic cells, they are --nucleotides long. |
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Definition
1,000 to 2,000 bacteria
150 to 200 EUK |
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Term
DNA polymerase alpha=
DNA polymerase delta=
which form is for prok |
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Definition
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Term
| Origin of replication: Prokaryotes vs. eukaryotes |
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Definition
• Bacteria: single origin of replication
• Eukaryotes: multiple specific sites at which replication is originated |
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Term
| Why multiple sites of origin in EUK? |
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Definition
| • Because eukaryotic cells contain much more DNA than bacteria, there must be multiple origins of replication on each chromosome in order to replicate all of the DNA in a timely fashion |
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Term
| One origin of rep in bacteria = |
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Definition
| begins at the same spot on the chromosome every time |
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Term
E. coli site of origin of rep =
what is the seq? |
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Definition
In E. coli, this site is called OriC ("origin of chromosome replication")
• OriC is a 9 base-pair (bp) AT-rich sequence that is repeated 4 times within the region |
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Term
| Humans have a large genome --bp and an estimated -- replication origins. Replication in humans takes about -- hours. |
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Definition
(~3 billion bp)
10,000
10 hrs |
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Term
| Problem of replication in bacteria: |
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Definition
| Start at ori of rep only one site on circular dna- goes in two direction until it meets at the bottom of the circle opposite of the site of origin. |
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Term
| The initiation of DNA replication is mediated by |
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Definition
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Term
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Definition
| , a protein that binds to a region of the origin known as the DnaA box |
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Term
| In E. coli, there are # DnaA boxes, each of which contains |
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Definition
5
a highly conserved 9 bp consensus sequence 5' - TTATCCACA - 3'. |
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Term
| Binding of DnaA to this region causes it to become |
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Definition
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Term
| Following DnaA binding, a region of OriC upstream of the DnaA boxes |
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Definition
| (known as DnaB boxes) become melted. |
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Term
| Melting of the DnaB boxes requires -- |
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Definition
| ATP (which is hydrolyzed by DnaA). |
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Term
Following melting, DnaA recruits --
this forms-- |
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Definition
| a hexameric helicase (six DnaB proteins) to opposite ends of the melted DNA. This is where the replication fork will form. |
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Term
| . The binding of dNTPs causes a |
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Definition
| conformational change which allows the dnaB to translocate along the DNA, thus mechanically forcing the separation of the DNA strands. |
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Term
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Definition
| also known as the helicase loader, is a regulator of DnaB |
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Term
DnaA binds at the
Pulls apart--
The origin of replication is
-- provides energy for -- |
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Definition
origin of replication
the 2 strands of the dsDNA
AT-rich
ATP for melting |
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Term
| Strand separation continued by -- = -- |
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Definition
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Term
| DnaB binds to -- and is a -- |
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Definition
opposite ends of separated dsDNA (i.e., 1 helicase molecule per replication fork)
Hexamer (= 6 subunits) |
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Term
| Lagging strand of parental dsDNA runs through the |
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Definition
| central channel of helicase |
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Term
| --required for loading of DnaB hexamer |
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Definition
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Term
| Ori of rep has an abnormally high AT content bc |
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Definition
| melting point is lower and you only have to seperate two H bonds instead of 3. |
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Term
| helicase works by running through |
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Definition
| lagging strand to keep strands seperated |
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Term
| What keeps strands from supercoiling induced by seperating the strands of helical structue? |
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Definition
| DNA gyrase attaches ahead of replication fork |
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Term
| dna wants to avoid a-holes) Avoids them by |
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Definition
| topoisomerase 2(cleaves both strands, turns them, reforms them)=gyrase runs ahead rep form and relaxrd positive supercoils |
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Term
| Leading strand is excluded from |
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Definition
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Term
| ergy from ATP used to provide |
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Definition
| mechanical energy to translocate along the DNA |
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Term
| Unwinding of parental dsDNA introduces |
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Definition
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Term
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Definition
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Term
| DNA strand separation maintained by -- via |
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Definition
single-strand binding protein (SSB)
• Maintains ssDNA in a linear form (instead of spontaneous kinks) |
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Term
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Definition
| segment with high AT content the DNAA binds and puts tension on DUE segment making it unwind- 2 rep forks. |
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Term
| # of helicase per replcation site |
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Definition
| 2- one going each way on lagging strand only |
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Term
| Formation of the helical DnaA complex is facilitated by the proteins |
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Definition
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Term
| DNA-unwinding elements (DUEs) are |
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Definition
| are specific base sequences that are located in the origin of DNA replication where they provide the start point for strand separation and unwinding of the DNA double helix. |
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Term
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Definition
recognize ori sequence; open duplex at specific sites
1 subunit |
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Term
| DnaB protein function (helicase)- |
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Definition
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Term
Primase (dnaG protein) function-
subunits- |
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Definition
synthesizes RNA primers
4 subunits |
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Term
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Definition
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Term
DNA gyrase (DNA topoisomerase 2) function-
subunits- |
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Definition
relives torsional strain generated by unwinding
4 |
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Term
| At the rep fork one nt comes in as -- and -- provide energy to make -- |
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Definition
| One nucleotide comes in as the dNTP- triphosphates provide energy for making the covalent bond. |
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Term
| RNA polymerase can -- but needs -- |
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Definition
| start with first nt (does not need primer) Has to have a template (DNA) |
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Term
| Primase makes --nt of rna primer before |
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Definition
10 nt
then DNA polymerase 3 then takes over synthesising DNA adding on to the primer. |
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Term
| Primase is -- dependent rna polymerase |
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Definition
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Term
| Leading strand rep fork moves -- on the --template |
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Definition
| 3’ 5’ on the lagging strand template |
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Term
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Definition
| discontinuous synthesis in 5’ 3’ direction. Must read template 3’ 5’ while running along it in 5’ 3’ direction. |
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Term
| 2 problems of lagging strand- |
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Definition
| 1. Rna must be removed and replaced with DNA. 2. A polymerase cannot close the gap (join the two fragments) |
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Term
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Definition
| (5’ 3’ exonuclease + DNA polymerase activities) |
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Term
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Definition
| ) binds at the gap “nick” and runs 5’ 3’ (wrong direction) |
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Term
| DNA polymerase 1 exonuclease activity- |
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Definition
| exonuclease activity= takes off nt from the end. |
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Term
| DNA polymerase 1 enzyme activities: |
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Definition
| Has 3 enzyme activities= hydrolys off the ribo nt off primer and replace it with a deoxy nt and as it does it converts rna to dna. When it gets to the nick it cannot make the phospho diester bond, req the DNA ligase enzyme reqs energy (atp) |
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Term
| Why does DNA polymerase req a primer? |
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Definition
| needs pre-existing 3' OH group to attack the alpha P atom of the incoming dNTP |
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Term
| RNA polymerase inserts first nt into the - end of new rna strand |
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Definition
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Term
Primase has -- rxn compared to DNA polymerase.
Differences: |
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Definition
SAME RXN, except:
• Uses ribonucleotides (NTP), not dNTP
• No primer required |
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Term
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Definition
two nucleotides hydrogen-bonded to consecutive bases in the same template, but not covalently bonded to each other via a phosphodiester bond
Uses ATP (or NAD+) as energy source |
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Term
| DNA polymerase 1 3enzymatic activites: |
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Definition
5→3 polymerase activity (DNA synthesis)
3→5 exonuclease activity (proofreading)
5→3 exonuclease activity (removes the RNA primer |
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Term
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Definition
| (= ave. # of nucleotides added before enzyme dissociates from template) |
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Term
Processivity of DNA poly 1=
rate of rxn= |
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Definition
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Term
Processivity of DNA poly 3=
rate of rxn= |
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Definition
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Term
| key player in replication, most active, but lease abundant in cell= |
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Definition
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Term
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Definition
5→3 polymerase activity (DNA elongation)
3→5 exonuclease activity (proofreading) |
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