Chapter 25 Exam 2 Flashcards
How are bacterial genes named?
Genes typically named using three italicized lowercase letters reflecting a function
examples: dna, uvr, and rec
capital letters added to abbreviation reflect order of discovery, not enzymatic order
examples: dnaA, dnaB, and dnaQ
How are bacterial proteins named?
Proteins often named after their genes using nonitalicized, roman type with the first letter capitalized
examples: DnaA (encoded by dnaA) and RecA (encoded by recA)
How are eukaryotic genes named?
*no single convention exists for all eukaryotic systems
*in Saccharomyces cerevisiae, gene names are three italicized uppercase letters followed by an italicized number
example: COX1
How are eukaryotic proteins named?
Protein naming is complex and variable
in yeast, some proteins have long common names
example: cytochrome oxidase
other yeast proteins have the same name as the gene, with one uppercase and two lowercase letters in roman type, followed by a number and the letter “p”
example: Rad51p (encoded by RAD51)
How is DNA replication semiconservative?
semiconservative replication = each DNA strand serves as a template for the synthesis of a new strand
produces two new DNA molecules, each with one new strand and one old strand
established by Meselson and Stahl in 1957
What is a replication fork?
replication forks = dynamic points where parent DNA is being unwound and separated strands replicated
both DNA strands are replicated simultaneously
both ends of the bacterial chromosome have active replication forks (bidirectional replication)
What is a denaturation map and what does it show?
denaturation mapping = selective denaturing of sequences unusually rich in A=T base pairs to provide landmarks along the DNA molecule
generates a reproducible pattern of single-strand bubbles
origin = location where replication loops are initiated
*It shows that replication loops always initiate at an origin
How does DNA synthesis proceed?
DNA Synthesis Proceeds in a 5′→3′ Direction and is Semi-discontinuous
*new DNA strands are always synthesized in the 5′ → 3′ direction
the free 3′-OH serves as the point of elongation
What are Okazaki fragments?
Okazaki fragments = short DNA fragments that are synthesized in the replication of one of the new DNA strands
~150 to 200 nucleotides long in eukaryotes
~1,000 to 2,000 nucleotides long in bacteria
spliced together by DNA ligase
What is a leading strand?
leading strand = 5′ → 3′ synthesis proceeds in the same direction that the replication fork moves
synthesized continuously
What is a lagging strand?
lagging strand = 5′ → 3′ synthesis proceeds in the opposite direction that the replication fork moves
synthesized discontinuously through the synthesis of Okazaki fragments
*new DNA strands are always synthesized in the 5′→3′ direction
*the template is read in the 3′→5′ direction
What are nucleases?
nucleases = enzymes that degrade nucleic acids
DNases = specifically degrade DNA
What are exonucleases?
exonucleases = degrade nucleic acids from one end of the molecule
many operate in only one direction (either 5′→3′ or 3′→5′)
What are endonucleases?
endonucleases = degrade nucleic acids at specific internal sites in the molecule
What is DNA polymerase?
DNA polymerase = complex enzymes that can synthesize DNA
-many have additional activities
-E. coli cells have at least 5 DNA polymerases, including DNA polymerase I
*the general reaction for DNA polymerization is:
(dNMP)n + dNTP → (dNMP)n+1 + PPi
where dNMP and dNTP are a deoxynucleoside 5′-monophosphate and 5′-triphosphate, respectively
What is the DNA polymerase reaction?
the reaction is a phosphoryl group transfer
the 3′-OH of the nucleotide at the 3′ end of the strand (the nucleophile) attacks the α phosphorus of the incoming dNTP
one Mg2+ ion helps deprotonate the 3′-OH group
makes it a more effective nucleophile
Other Mg2+ ion binds the incoming dNTP and facilitates departure of PPi
What does DNA polymerase require?
DNA Polymerases Require a Template and a Primer
polymerization is guided by a template DNA strand according to Watson and Crick base-pairing rules
primer = a strand segment with a free 3′-OH group to which a nucleotide can be added
must be complementary to the template
many are RNA oligonucleotides
primer terminus = the free 3′ end of the primer
What is an insertion site?
= the portion of the active site where the incoming nucleotide binds
One of the active sites of DNA polymerase
What is the postinsertion site?
the portion of the active site where the new base pair resides when the polymerase slides forward
One of the active sites of DNA polymerase
What is processivity in regards to DNA polymerases?
processivity = the average number of nucleotides added before a polymerase dissociates
DNA polymerases vary greatly in processivity.
ranges from a few nucleotides to many thousand
How accurate is replication?
errors in E. coli occur every 1 in 109 to 1 in 1010 nucleotides added
*equates to an error only once per 1,000 to 10,000 replications
*the active site of DNA polymerase excludes base pairs with the incorrect geometry
*DNA polymerases insert one incorrect nucleotide for every 104 to 105 correct nucleotides
-may occur because a base is in a tautomeric form
Contribution of Base-Pair Geometry to the Fidelity of DNA Replication
How does error correction happen in DNA replication?
Error Correction by DNA Polymerase I
translocation of the enzyme is inhibited when an incorrect nucleotide is added
many DNA polymerases have intrinsic 3′→5′ exonuclease proofreading activity
permits the enzyme to remove a newly added nucleotide
What is proofreading?
proofreading = DNA polymerase activity that involves replacement of the incorrect nucleotide
requires the expenditure of three high-energy bonds
proofreading improves the accuracy 102 - to 103-fold
one error in every 106 to 108 bases remains after base selection and proofreading
the higher measured accuracy of E. coli replication comes from mismatch repair
What is DNA polymerase I in E.coli and what is its function?
polA, proofreading, has 3-5 prime and 5-3 prime exonucleases (1 subunit)
DNA polymerase I is abundant, but insufficient for replication of the E. coli chromosome
rate (600 nucleotides/min) is slower than observed for replication fork movement
low processivity
the primary function of DNA polymerase I is cleanup during replication, recombination, and repair
What is the function of DNA polymerase II (E.coli)?
polB, proofreading (3-5 prime), 7 subunits
Involved in DNA repair
What is DNA polymerase III in E.coli and what is its function?
polC (dnaE) proofreading (3-5 prime), 9 subunits
The principal replication enzyme in E.coli
What is DNA polymerases IV and V in E.coli and what are their functions?
dinB (1 subunit), umuC (3 subunits) no proofreading
Involved in an unusual form of DNA repair
How does polA show exonuclease activity differently?
the 5′→3′ domain is in front of the enzyme and performs nick translation
nick translation = a break or nick in the DNA is moved along with the enzyme
important in:
DNA repair
the removal of RNA primers during replication
mild protease treatment separates this domain from the remainder of the enzyme (the large fragment or Klenow fragment) retains the polymerization and 3′ → 5′ proofreading activities.
What is nick translation?
nick translation = a break or nick in the DNA is moved along with the enzyme
important in:
DNA repair
the removal of RNA primers during replication
(When the 5′→3′ exonuclease domain is removed, the remaining fragment (𝑀r 68,000), the large fragment or Klenow fragment, retains the polymerization and proofreading activities.)
What are the subunits of Polymerase III?
9 subunits
Theta or θ subunit associates with α and ε subunits to form a core polymerase
can polymerize DNA but with limited processivity
up to three core polymerases are linked by a clamp-loading complex (τ3δδ′)
How does β Subunits convert DNA polymerase III* to DNA Polymerase III Holoenzyme?
DNA polymerase III* = the entire assembly of 16 protein subunits (eight different types)
β subunits = provide an increase in processivity by preventing dissociation of DNA polymerase III from DNA
one dimer of β subunits associates with each active core subassembly
addition of β subunits converts DNA polymerase III* to DNA polymerase III holoenzyme
What is DNA replicase system?
DNA replicase system (replisome) = the entire complex of enzymes and proteins required for replication in E. coli
consists of 20+ different enzymes and proteins
*helicases, topoisomerases, DNA-binding proteins, primases, DNA poly I, RNase H1, DNA ligases
What is are helicases?
helicases = enzymes that move along the DNA and separate the strands
requires chemical energy from ATP
enzyme
What are topoisomerases?
topoisomerases = relieve topological stress created by strand separation
enzyme
What are DNA-binding proteins?
DNA-binding proteins = stabilize separated strands
enzyme
What are primases?
enzymes
primases = synthesize short segments of RNA to serve as primers
What is DNA polymerase I?
enzyme
DNA polymerase I = removes and replaces RNA primers
what are RNase H1?
Enzyme
RNase H1 = a specialized nuclease that degrades RNA in RNA-DNA hybrids
can remove RNA primers
What are DNA ligases?
Enzyme
DNA ligases = seals nicks in the DNA backbone following removal and replacement of an RNA primer
What are the three stages of the E.coli chromosome replication?
DNA synthesis can be divided into three stages:
initiation
elongation
termination
What is initiation of the E.coli chromosome?
*the E. coli replication origin (oriC) consists of:
five repeats of a 9 bp sequence (R sites) that serve as binding sites for the key initiator protein, DnaA
*the DNA unwinding element (DUE) = a region rich in A=T base pairs
*three additional DnaA-binding sites (I sites)
*binding sites for the proteins IHF (integration host factor) and FIS (factor for inversion stimulation)
What are some of the proteins required for initiation at the E.coli origin? (10 proteins)
DnaA protein- recognizes oriC sequence; opens duplex at specific sites in origin
DnaB protein (helicase)- unwinds DNA
DnaC protein- required for DnaB binding at origin
HU- histonelike protein; DNA-binding protein; stimulates initiation
FIS and IHF- DNA binding proteins that stimulate initiation
Primase (DnaG protein)- synthesizes RNA primers
Single-stranded DNA binding-protein (SSB)- binds single-stranded DNA
DNA gyrase (DNA top II)- Relives torsional strain generated by DNA unwinding
DNA methylase- methylates 5’ GATC sequence at oriC
What is the role of Dna A proteins?
DnaA Proteins Bind at R and I Sites in oriC
eight ATP-bound DnaA molecules bind to the R (5) and I (3) sites in oriC
strand separation in the A=T-rich DUE occurs due to:
strain due to positive supercoiling
binding of DnaA to the DUE region
*DnaA has a higher affinity for R sites than I sites, and it binds R sites equally well in its ATP- or ADP-bound form. The I sites, which bind only the ATP-bound DnaA, allow discrimination between the active and inactive forms of DnaA.
How does DnaB helicase get loaded?
DnaC protein = an AAA+ ATPase that loads DnaB protein onto the separated DNA strands
-two ring-shaped DnaB hexamers are loaded in the DUE, one onto each DNA strand
DnaB protein (helicase) = migrates along ssDNA in the 5′→3′ direction and unwinds DNA
What is the role of SSB and DNA gyrase in initiation?
single-stranded DNA-binding protein (SSB) stabilizes separated strands
DNA gyrase (DNA topoisomerase II) relieves topological stress ahead of the replication forks
Why does initiation only occur once in each cell cycle?
Hda protein = an AAA+ ATPase that binds to the β subunits of DNA polymerase III and stimulates hydrolysis of the ATP bound to DnaA
-causes disassembly of the DnaA complex
homologous to DnaA
release of ADP by DnaA and rebinding of ATP occurs on a time scale of 20 to 40 minutes
How is replication regulated by methylation?
Dam (DNA adenine methylation) methylase = methylates the N6 position of adenine within the palindromic sequence (5′)GATC of oriC DNA
hemimethylated oriC sequences are sequestered by:
interaction with the plasma membrane
binding of the protein SeqA
after SeqA dissociation, oriC sequences are released from the membrane and Dam methylase fully methylates DNA (both strands) to allow new DnaA to bind
How is the leading strand elongated?
the more straightforward of the two strands
primase (DnaG) synthesizes a short (10 to 60 nucleotide) RNA primer at the replication origin
requires interaction with DnaB helicase
Primase and DnaB move in opposite directions
DNA polymerase III adds nucleotides to the 3′ of the primer
linked to the DnaB tethered to the opposite DNA strand
How is the lagging strand elongated?
accomplished in short Okazaki fragments
as in leading strand synthesis, primase synthesizes an RNA primer and DNA Pol III adds nucleotides to the 3′ end
DNA of the lagging strand loops around so one asymmetric DNA Pol III dimer complex can synthesize both strands
How are the leading and lagging strands synthesized by DNA Poly III?
DNA polymerase III uses one set of its core subunits to synthesize the leading strand continuously
the other two sets of core subunits cycle from one Okazaki fragment to the next on the looped lagging strand
What is the first step of the leading and lagging strand sythesis?
DnaB helicase travels along the lagging strand template in the 5′→3′ direction and unwinds the DNA
DnaG primase occasionally associates with DnaB helicase and synthesizes a short RNA primer
What is the second step of leading and lagging strand synthesis?
a new β sliding clamp is positioned at the primer by the clamp-loading complex of DNA polymerase III
What is the third step in the synthesis of the leading and lagging strand?
replication halts after synthesis of an Okazaki fragment is completed and DNA polymerase III core subunits dissociate from the β sliding clamp
How does recombination in meiosis take place?
The DNA strand invasion in eukaryotes is catalyzed by RecA-like recombinases called Rad51 and Dmc1. Loading of Rad51 onto DNA is promoted by Rad51 loading protein BRCA2 (analogous to the bacterial RecF, RecO, and RecR proteins).
What is the process of NHEJ?
(DNA Ligase) Small DNA gaps are filled by a DNA polymerase, Pol m or Pol l .
the nicks are sealed by a protein complex consisting of XRCC4 (x-ray cross complementation group), XLF (XRCC4-like factor), and DNA ligase IV.
What is the process of Site-specific recombination(with Tyr)?
Site-Specific Recombination with Tyr Recombinase Enzymes
the recombinase is covalently linked to the DNA at the cleavage site
cleaved DNAs are rejoined to form a Holliday intermediate
exchange is reciprocal and precise
We can view a recombinase as a site-specific endonuclease and ligase in one package.
What are effects of site-specific recombination?
outcome depends on the location and orientation of the recombination sites in a dsDNA molecule
What is the XerCD system?
resolution of a Holliday intermediate can generate a contiguous dimeric chromosome
XerCD = a specialized site-specific recombinase in E. coli
converts the dimer to monomers
allows chromosome segregation and cell division to proceed
How are transposon’s inserted?
short repeated sequences at each end of the transposon serve as binding sites for the transposase
become duplicated following transposon insertion
What are two general pathways for transposition?
direct transposition = “cut and paste”
replicative transposition = “copy and paste”
How does recombination of IgM genes happen?
during formation of a mature B lymphocyte, one V segment (first 95 aa) and one J segment (remaining 12 aa) are brought together
40 x 5 = 200 possible V–J combinations
additional variation at the V-J junction increases this to 500
Recombination of IgM genes?
Fig depicts the organization of the DNA encoding the kappa light chains of human IgG and shows how a mature kappa light chain is generated.
The V (variable) segment encodes the first 95 amino acid residues of the variable region, the J (joining) segment encodes the remaining 12 residues of the variable region, and the C segment encodes the constant region.
What is the mechanism of recombination of IgM genes?
recombination signal sequences (RSS) = found between the V and J segments
RSS bind to proteins RAG1 and RAG2.
RAG = products of the recombination activating gene
RAG proteins catalyze formation of a double-strand break between segments to be joined
