Molecular Biology and Biotechnology
Test 2 Spring 2006
Catalytic RNA is RNA that exhibits enzymatic behavior. It can be RNA alone of RNA
associated with proteins.
Groups I, II, III: RNA only. Self-splicing introns seen in prokaryotes only.
Ribonuclease P is protein plus RNA. It processes and cuts to correct size the
tRNA.
Bacterial Ribosomes: RNA plus proteins; form peptide bonds.
DNA Replication
Parent DNA is opened up; daughter DNA is built onto each strand; result is 2 DNA, each
with one old and one new strand of DNA.
1. Replication fork formed: parent strands are separated by Helicase (“melting”).
SSB (single stranded binding protein holds apart two separated strands of SS DNA) holds
strands open. In Eukaryotes, SSB is replaced by RPA, replication protein A.
2. Replication:
a. Primer, a strand of RNA, is added to the opened DNA strand by DNA-Gprimase in prokaryotes and Pol-alpha in eukaryotes. Primer provides a 3’ end for
the polymerase to add bases to. Necessary to start replication.
b. A polymerase adds nucleotides to the 3’ end of the primer. Prokaryotes use Pol
III and eukaryotes use Pol delta. Pol transports Nucleotide triphosphate to
primer/DNA strand. It attaches; pyrophosphate is removed by nucleophilic attack
(OH on ribose attacks phosphate).
c. Proofreading is done by the same Pol – after each base that it adds, it reviews
the base. If it is the wrong base, Pol exhibits 3’-5’ endonuclease activity and cuts
the base out.
3. Polarity: A problem arises when replicating the 5’-3’ parent strand of DNA because
Pol must add bases to a 3’ end. The replication fork mentioned above opens the DNA up,
exposing two strands of DNA: one is 5’-3’ and one is anti-parallel: 3’ - 5’. Replicating
the 3’ -5’ strand is easy; since bases are added anti-parallel, the new DNA adds 5’-3’,
leaving the 3’ base available for Pol to add the next base to. This replication can proceed
easily and continuously.
However, in the 5’ – 3’ strand (known as the discontinuous strand), the anti-parallel new
DNA that is being formed would only have 5’ bases available to attach. Pol can only add
to 3’, so instead is has to work backwards in small pieces. As the fork opens, primer is
added and Pol works 5’ to 3’ to add bases. Many primers are added and fragments of
DNA synthesized as the fork opens. These fragments are called Okazaki fragments.
4. Clean up: after nucleotides are added, the primers must be removed (or else we’d have
random RNA mixed into our DNA).
RNAse H removes primers but leaves gaps: spaces that are missing nucleotides. Gaps are
filled by Pol I in prokaryotes and Pol delta in eukaryotes: adds the correct nucleotide.
This still leaves a nick (missing a phosphodiester bond). It is repaired by ligase. There are
many gaps in the discontinuous strand because each Okazaki fragment has its own
primer.
Ligase mechanism: Ligase contains a tyrosine that is “loaded” with an ATP. Loss
of a phosphate results in AMP; AMP is transiently transferred to the nucleotides
that have a nick between them. Nucleophilic attack removes a phosphate and
release AMP, leaving the phosphate bond.
Enzyme complex: In order to replicate efficiently, DNA strands are pulled through a
donut (yum!)-like protein complex that keeps all the enzymes together and ensures that
replication occurs in an orderly fashion. How it works: A protein complex called a sliding
clamp in prokaryotes or PCNA (Proliferating Cell Nuclear Antigen) in eukaryotes circles
the DNA strand; as replication progresses enzymes are held in place. This occurs
simultaneously on BOTH strands of DNA, and the two protein complexes are held
together in a single unit known as a replisome. This forces the replication to progress at
the same pace to prevent the continuous strand from outpacing the discontinuous strand
Unique to Eukaryotes: more complex cells have more complex replication. For example,
when the donut complex is assembled on DNA in eukaryotes, it requires RCF, replication
factor C, which puts the clamp in place, then disassembles. Eukaryotes also require FEN1, flap endonuclease 1, which removes the last nucleotide (“flap”) from the primer.
In addition, while prokaryotes generally have only 1 ORI site, eukaryotes have many.
Replication can occur simultaneously at several adjacent sites called replicons that merge
together. To form the replicon, first the ORC, origination recognition complex, finds the
ORI site and starts forming replicon. MCM, mini-chromosome maintenance proteins, are
first parts of replicon. They begin to separate strands in order to allow helicase to work.
Prokaryotes vs. eukaryotes: DNA replication
Splits
Holds adds
Adds
Removes
ds DNA split
primer
nucleotides
primer
DNA
to build DNA
open
strand
Prokaryotes Helicase SSB
DNA-G- Pol III
RNAse H
Primase
Eukaryotes Helicase RPA Pol-alpha Pol delta
RNAse H
(Continued)
Repairs
“donut” clamp Puts clamp in place
nick
Prokaryotes
ligase
Sliding clamp
NONE
Eukaryotes
ligase
PCNA
RCF
(Continued)
Identifies ORI First part of
replicon
Prokaryotes
NONE
No replicon!
Eukaryotes
ORC
MCM
Fills in
gaps
Pol I
Pol delta
Removes Flap
NONE
FEN-1
Telomeres
The discontinuous strand runs into a problem at the end of DNA: a primer has to bind so
the last Okazaki fragment can be made, but there is no place to add the primer. Telomeres
are extra pieces of DNA (TTAGG repeats) that give a place for primer to bind – without
telomeres, the DNA would gradually shorten because the last few BP’s would not be
duplicated. Teleomeres do not code for genes and, due to repeats, tend to fold into nonBDNA quadroplexes. With time, the DNA still shortened and more telomeres must be
added.
Telomerase are RNA and protein. The RNA is the template for the sequence that it adds
to the SS 3’ end only (sequence is part of the enzyme – unique). Because it adds to 3’
end, this strand is 18 nucleotides longer than the other.
Cross-Over
1. Homologous: chromosomes from each parent may exchange homologous pieces of
DNA.
2. Non-homologous: Natural exchange of non-identical sequences.
Transposons are long sections of junk DNA that do not code for DNA but do code
for the enzyme transposase. Transposase, if expressed, cuts out the transposon and
relocates it in a random location on the DNA.
Mobile Elements do not code for an enzyme; they move randomly around the
genome by an unknown mechanism. LINE or SINE (long or short interspersed
mobile elements).
Non-homologous chromosomes can be very damaging to DNA by disrupting gene
sequences.
Carcinogens initiate neoplastic cells.
Mutagens are carcinogens that damage DNA – UV, X rays etc.
Promoting compounds such as hormones or mitogens (stimulate cell division) can cause
tumor growth.
Steps to carcinogenesis:
1. Initiation: formation of a neoplastic (transformed, abnormal) cell. Typically involves
DNA damage. Usually neoplastic cells are destroyed by apoptosis; if not, they can
become cancerous.
2. Promotion: Rapid division of neoplastic cells forms tumor (if encapsulated, considered
benign). Cell division caused by a promoting compound.
3. Progression: Rapid, unchecked growth; invades surrounding tissue.
4. Metastasis: Invades other types of tissue.
Treatment usually begins at step 3 or 4.
Types of Carcinogens:
A complete carcinogen acts as an initiator and a promoter.
Genetic: damage DNA by base alterations, ss breaks, or ds breaks to alter
coding/regulatory sequence. Possible alterations include:
- activate a proto-oncogene
- deactivate a tumor-suppressor gene
- deactivate a DNA repair mechanism
Epigenetic: Regulate cell growth. Can mimic stimulatory hormones or induce growth
factors.
Cell Cycle and Cancer
Cells are most susceptible to damage during s or m phase. Therefore, rapidly dividing
epithelial cells are susceptible to carcinogens. Epigenetic carcinogens can transform
quiescent (gap phase – non-proliferating) cells and transform them into susceptible
proliferating cells.
Treating cancer: Most recent advances are in the areas of prevention, detection, and
surgery. Chemotherapy is costly and painful and generally only prolongs survival, rather
than conquer the cancer. More research in this area is needed. Combined treatments have
an additive effect.
Chemotherapy:
1. Anti-metabolites are nucleoside analogs that are incorporated into your DNA
This example is cytosine arabinoside, which is a pyrimidine metabolite used in leukemia
treatments:
2. Alkylating agents add alkyl groups; damage DNA to the extent that it cannot be
replicated.
3. Microtubule inhibitors interfere with microtubule activity necessary for cell division.
Taxotere (brand name is Taxol) is in this category.
Spontaneous DNA damage
1. Deamination: spontaneous non-liver microsomal metabolism results in deamination.
Usually repaired by the body.
2. Base removal: sometimes a base, usually a purine, is spontaneously removed, leaving
an “AP site” (apurine site). AP site refers to any site missing base(s). The phosphates and
sugars remain.
3. Methylation: occasionally triggered by a carcinogen. Some methylation is natural
(used to distinguish parent from daughter strand).
4. Dimerization: Two bases dimerize. UV light causes T=T binding.
5. Oxidation: radiation forms HO* (pretend that’s a radical  )
Reaction:
H2O radiation________>
HO* + H*
+
In water, continues: H* + H2O 
H + O2
With Fe, Fenton reaction occurs: H+ + O2 + Fe  2 HO*
Result: one radiation event gives three hydroxyl radicals, capable of oxidizing DNA
bases.
DNA Mutations
1. Point mutation: One nucleotide replaced by another.
a. Missense: mutation changes codon; codes for a different amino acid
b. Nonsense: mutation causes a stop codon; protein is truncated.
2. Insert or delete: a nucleotide is added or left out; causes a frameshift (alters every
codon following the mutation)
3. Triplet expansion: areas of DNA with triplets are susceptible to accidental triplet
repeats. Excessive triplets cause many problems; they can alter receptors such as the
androgen receptor.
DNA repair mechanisms
1. Base Excision Repair removes a damages base. Useful when there is a small structural
damage to a base (oxidation; deamination – minor changes only). Steps to repair:
I. Remove damaged base with glycosylase (creates an AP site)
II. Remove sugar with AP-endonuclease (cuts at the 5’ end) or AP-lyase (cuts at
the 3’ end). (If repairing an AP site, repair starts here).
III. Fill in gap with a nucleotide by polymerase.
IV. Repair nick with ligase.
2. Nucleotide excision: removes the entire nucleotide to repair major damage such as
alkylation. Repair uses XP proteins. XP stands for Xeroderma Pigmentosum, a genetic
recessive disease caused by a mutation in any of seven XP repair proteins. The disease
increases susceptibility to skin cancer. Steps to repair:
I. Identify damaged nucleotide with XPC (recognizes damage and stops).
II. Follow with XPA : recognizes damaged nucleotide OR XPC; stops; becomes
nucleus of the XP complex that forms.
III. Form XP excision complex around XPA. The complex includes a large unit
called TF2-H (transcription factor 2-helicase) that exhibits helicase activity. This
unit contains two XP proteins, XPB and XPD, that open up DNA. RPA holds the
strands open.
IV. Remove oligonucleotide with two XP proteins:
XPE cuts about 25 nucleotides off the 5’ end.
XPG cuts about 5 nucleotides of the 3’ end.
V. Fill in gap; repair nick.
3. Mismatch repair: repairs an insert, delete, or mismatch. Relies on repair enzymes
ability to recognize an out of place base. How can it tell which half of the DNA has the
wrong base? If the repair enzymes see, for example, an A=G pairing, they need to know
if the A or the G is out of place. Since DNA is semi-conservative, it is reasonable to
guess that the parent strand is normal and the newly-synthesized daughter strand is
damaged. Therefore, the newer strand should be repaired.
How to identify the newer strand? Maintenance methylase is an enzyme that recognizes
hemimethylated (one side is methylated) DNA. Cytosine in DNA is naturally methylated
at the 5-position by methylase, but this takes awhile after it the strand is made. Therefore,
if only one strand is methylated, it is the parent strand and the daughter strand has not yet
been methylated. This strand will be repaired.
Steps to repair:
I. Identify damage: Mut-s-alpha recognizes mismatch
Mut-s-beta recognizes insert or delete
II. Remove oligonucleotide with Mut-L- alpha plus an unidentified enzyme. Cuts
out several hundred nucleotides.
III. Fill in gap; replace nick.
Note: A methylated cytosine area of DNA is not transcribed, so methylation also acts as
replication marker. Therefore removing methyls can have two bad consequences: genes
that should not be transcribed will be transcribed and healthy parent strand DNA may be
wrongly “repaired” rather than damaged daughter strands.
Enzyme de-methylation: MGMT, Methyl Guanidine Methyl Transferase, removes
alkylations from bases (usually guanidine). Methylated guanine can bind to thiamine and
cause mutations. The enzyme’s active site contains cysteine; the sulfur removes the CH3.
4. Daughter Strand Gap Repair: During replication, polymerase can skip damaged areas
of the parent strand. This usually causes apoptosis. The body also has several not-soeffective repair mechanisms (these are ss repairs; major repairs can only be performed on
ds DNA so it is better to repair DNA before replication):
a. Recombination:
Polymerase replaces the damaged area of parent DNA with a homologous strand
of DNA, then replicates this template to make daughter DNA. Problem: if
homologous strand is not correct, results in mutations.
b. Bypass Synthesis:
Polymerase is forced top replicate past the damaged area to avoid a gap on
daughter strand. Problem: Often ends up being the wrong base; results in
mutations.
Strand Breaks
1. SS breaks caused by radiation or by Topoisomerase I inhibitors (Topo I cuts one
strand; if stopped at that point, have an SS break). Blocking Topo I prevents
transcription; replication.
- If Topo I remains attached, break cannot be repaired.
- If Topo removed, can repair with a ligase. However, this DNA is now available for
transcription because Topo I is no longer twisting it up. This can cause problems.
2. DS breaks caused by radiation or Topo II inhibitors. DS breaks cause major damage;
usually lethal to the cell. Stimulates a lot of recombination, increasing chance for a
mutation.
DNA repair basics:
- Better to repair before transcription
- Major repair can only be done on DS DNA
- Small amount of damage repairable
- Major damage overwhelms the repair mechanisms
- Bacterial DNA repair: damaged DNA initiates SOS system: genes for repair
enzymes are expressed, these enzymes are produced and repair DNA (I guess if
the damage is on the part that codes for repair enzymes, the po’ little bacterium
just dies).
Critical Periods of Development:
1. Embryonic/fetal development: Cell damage can cause miscarriage.
Oocytes develop from primary to secondary oocytes to ovums. Primary oocytes are
produced prenatally. Therefore, a woman’s exposure to a mutagen can affect her unborn
daughter’s DNA AND her unborn daughter’s primary oocytes: damage to the primary
occytes can result in damage to the woman’s grandchildren.
2. Organ development: Glandular organs are especially susceptible; for example, some
glands have maturation stages that make them resistant to damage. Mammary glands
have two such stages, puberty and parturition (child birth). If both of these stages are
reached, the gland is less susceptible to DNA damage. Populations less likely to have
children have more incidence of breast cancer ( nuns have higher breast cancer rates than
others).