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Eukaryotic DNA replication
The replication of eukaryotic DNA is more complex than that of microbial systems. Along with an increased amount of DNA there is also the additional complicity of histones associated with the duplex DNA strand.
As we have seen in microbial systems there are a variety of proteins associated with what one might call the replication complex which travels at the replication fork. In the eukaryotic cells there is considerable evidence that we also have what might be termed a replication complex. More correctly we will find that this should be plural, that is, that there are several replication complexes and that the replication of chromosomal DNA in the nucleus of eukaryotic cells is carried out by many replication complexes in a spatially regulated pattern on many sub-chromosomal units. The types of proteins included in such replication complexes include such things as:
Many specific examples of each of these proteins are mentioned in the literature concerning eukaryotic replication. Refer to the literature for various characteristics of the polymerases. The activities of the other enzymes are similar to the activity of the microbial proteins but do have predictable variation as far as specific individual characteristics in eukaryotic systems.
As mentioned, the replication of eukaryotic DNA occurs in specially arranged units which have been termed replication units (RU's). RU's are grouped in tandem on the chromosomes and may vary in length from 4 microns (8x106daltons, 1.3x104 base pairs) to as long as 280 microns (560 x 106 daltons, 90 x 104 base pairs). Most RU's, however, range in length from 10 to 100 microns. These relative sizes have been determined from eukaryotic microbes such as yeast, higher plants, vertebrates, and invertebrates. As mentioned RU's are tandemly arranged on the chromosome in clusters. These clusters may vary from as few as two RU's per cluster to as many as 250 RU's per cluster. The larger number of RU's per cluster appear to be as such examples as satellite DNA's which would include the repeating gene units for ribosomal RNA and for histones. There is some evidence to suggest that in such organisms as yeast the RU clusters may constitute the entire DNA strand of a single chromosome. In mammalian cells the clusters of RU's may represent a single chromosome band.
It appears that DNA synthesis is initiated at an origin site within the RU and usually proceeds bidirectionally by the activities of two replication forks synthesizing to opposite termini. That is to say that the origin does not necessarily have to be at one end or the other of the replication unit. There is some evidence, however, that certain short segments of DNA are replicated by a unidirectional fork movement in a variety of organisms. The proportion of bidirectional synthesis vs unidirectional synthesis is not known. However, most evidence suggests that much if not most DNA synthesis is in the bidirectional manner.
It appears that initiation sequences marking the origin site are located within DNA segments that contain palindromes. A palindrome is a sequence of bases which reads the same in both directions of a double helix. In essence there is an axis of two fold rotational symmetry. Interestingly enough this is also the basis for sight recognition for a variety of restriction endonucleases which are used in recombinant DNA research. The palindromes for origin recognition appear to be associated with AT-rich segments of DNA. In more detail, each individual replication unit regardless of the size is composed of a varying number of segments referred to as nucleosomes. Much of our knowledge of nucleosomes has been gained not only from isolation studies but also through the use of the electron microscope. In the relaxed chromatin material, small particles are seen. Depending upon the method of preparation these particles will range in size from 70 A in diameter to 125 A in diameter. Various names have been given to these particles in the literature such as nu-bodies. PS-particles, and finally nucleosomes. The latter, nucleosomes has been generally adopted. The nucleosomes have been found to be comprised of DNA plus histones. Each complete nucleosome is approximately 125 A in diameter. Within this 125 A particle there is a DNA segment, the precise conformation of which is unknown, of about 200 base pairs. Associated with this strand of duplex DNA, are five types of histones. H2A, H2B, H3 and H4 comprise a complex where each histone is represented in pairs. That is to say, we have an octomer of two pairs of each of these four histones, a total of 8 subunits. This octomer associates with about 140 base pairs of the DNA strand with the DNA wrapping around the outside of the octomer. This unit, which is approximately 110 A in diameter is referred to as the core particle. The remaining 60 base pair portion of the DNA strand is associated with one molecule of H1. This portion of the nucleosome is referred to as the linker. Together the core particle plus linker comprise the nucleosome. The number of nucleosomes per RU would of course vary according to the size of the RU.
The precise association of H2A, H2B, H3 and H4 are unknown and remains to be detailed with X-ray diffraction after this histone association is isolated in crystalline form. Furthermore the precise arrangement of the DNA on the surface of the histone complex is unknown. Also unknown is the role that these histones play in the association of the proteins of the replication complex with the DNA.
As a footnote, we should indicate there are a variety of names for each of the histones indicated above. H1 is also called F1 and simply I. H2A is referred to in some older literature as F2A2 and IIb H2B is referred to as F2B and IIb. H3 is referred to as F3 and III, while H4 is referred to often as F2A1 and IV. Also the molecular weights of the histones are as follows as determined with calf thymus histones. H1 = 21000, H2A 14000, H2B 13774, H3 15324 and H4 11282, all in kilodaltons.
Mammalian (and other eucaryotic) DNA Polymerases:
Five - so far a, b, g, d, e
Pol a
Probably nuclear = tetramer - 160-180 kd catalytic + 70-75 kd
+ 2 primase peptides of 55-60 kd and 48-50 kd.
calf thymus = 165 kd + 68 kd + primase + calmodulin binding protein
yeast is similar to calf thymus
Most abundant of all polymerases in eucaryotes. Amount is proportional
to rate of proliferation. Found on leading strand during initiation
then on lagging strand thereafter.
75 kd subunit - function unknown - may link
catalytic unit to primase
- mcAb against this subunit inhibits primase but not catalytic
subunit.
- in Drosophila this peptide "covered up" a 3'-->5'
exonuclease activity associated with the large (catalytic) subunit.
- Special isolation techniques with calf thymus yielded a "holoenzyme"
with >10 peptides - including: polymerases, primases, 3'->5'
exo, calmodulin binding, DNA topoisomerase II, helicase act, methylase
act, and RNAse H.
3'-->5' Exo - not common, provisionally found in multiprotein complexes of calf thymus and HeLa cells.
Primase activity. - tightly bound to polymerase
peptide.
- yeast = 58 kd + 48 kd, 48 kd has ATPase act - thus probably
the primase active site.
- calf thymus = 55 or 59 kd + 48 kd, 48 kd = GTPase activity thus
is catalitic site, 59 Kd = "stabilizer"?
- mouse = 115 kd + 58 kd, 58 kd has primase act.
- yields RNA primer of 9-14 nucs long.
- Ideal for lagging strand replication of Okasaki fragments at
replication fork.
C1 and C2 proteins co-purify with multiprotein
complexes from HeLa cells.
-C1 = homo-tetramer (24kd each) + C2, a monomer of 52 Kd = C1C2
complex.
- increases primase activity by eliminating "non-productive"
binding of the polymerase complex - Does not affect processivity.
RNAse H - stimulates the polymerase activity
in vitro
- so far isolated from yeast & Drosophila
- 70 kd from yeast (monomer)
- from Drosophila = tetramer - 2 x 49 kd + 2 x 39 kd.
- probably stimulates by increasing the recycling of primers thus
increasing rate of initiation.
- Some studies suggest the need for an accessory
factor to ensure processivity. eg. mouse helix destabilizing protein-1
and AF-1 allowed processive replication of the M13 vial DNA. However,
calf thymus immunopurified (4 subunit) pola replicated M13 w/o
accessory factors.
-gene = pola - many homologous domains between species.
-in humans - 4 regions show highly conserved sequences with several
other species.
Pol b
Nuclear , amount does not vary with prolferation rate:
39 - 40 kd --> 335aa in mouse & human.
= 50% of activity in resting cells
= 5% of activity in rapidly dividing cells.
Size varies with species, 39-43 kd, no exonuclease activity, no primase activity, prefers activated DNA, shows low processivity and fidelity. Repair?? yup.
Pol g :
Pol d & e
- major component at replication forks.
- first isolated in 1976 from rabbit bone marrow cells
- possess a 3'-->5' exo ---> proofreading
- calf thymus - confusion in literature circa
1990 - 2 forms suggested
pold1
= 2 subunits 125kd + 48kd - PCNA increases processivity
pold2
= 2 forms 1 = 120kd + 40kd = processive w/o PCNA
2 = 5 subunits - also has primase activity
.- Discovered a pold then resolved it into 2 forms - pold1, & pold2, now pold1 is just pold while pold2 is now pole
-pold - not very processive alone - processivity is increased by the presence of proliferating cell nuclear antigen (PCNA)
-pole is highly processive by itself - no PCNA needed
- in yeast, 2 genes found, yielding the two forms. The 2 forms in mammals may be attributed to variations due to isolation techniques or to separate genes - not conclusive yet.
PCNA - First demonstrated as NA (nuclear antigen) in SLE auto-immunity. Also called "cyclin" but other molecules given this name by others. So ... since this is found only in proliferating cells it is called proliferating cell nuclear antigen or PCNA.
- homotrimer with each monomer about 2/3 the
size of prokaryotic b protein clamp.
- each monomer has 2 structural domains but combined, has same
topography as prokaryotic sliding clamp but with different DNA
sequence. Homology ??
- increases processivity of pold
- sequences for PCNA and on pol d, involved in their association with each other are highly conserved among species. eg. calf thymus PCNA can bind and increase processivity of yeast pol III (d1). Calf thymus pold, works with yeast PCNA (yPCNA) but needs 10X normal levels. Suggests more than one contact point between the two peptides.
3' - 5' EXO - yeast pol III (d1) degrades ssDNA to mononucleotides - sensitive to aphidicolin
- calf thymus pole can degrade small ssDNA (<6-7 bases) insensitive to aphidicolin w ssDNA template, very sensitive to aphidicolin with dbstDNA.
Pola and pold probably work together at the replication fork as a "hetero-holoenzyme dimer". d on the leading strand and d on the lagging strand during replication?
System has the usual binding, stabilizing,
unwinding, swiveling, gyrating and priming (RNA) proteins as did
the procaryotes.
All have similar but variable sizes.
INITIATION:
Occurs at origin sites of which there are numerous.
Timing of initiation at the sites must be coordinated so that
each RU is replicated once each turn of the cell cycle. An origin
recognition complex (ORC) containing about 6 peptides remains
bound throughout the cycle at "autonomously replicating sequences"
(ARSs). ARSs have a 11mer conserved sequence + 2-3 other shorter
conserved sequences within a 100-200 bp frame. It is suggested
that these proteins bind other regulatory proteins generated for
the S phase of the cell cycle.
A prereplication complex is suggested and cyclin dependent kinases
(Cdks) are involved with the timing of the S phase. [Cdks are
protein kinases that depend on the presence of cyclin to be active.
Regulation of the activity is via the degradation of the cyclin
subunit.]
Cdc2 and it's cyclin bind to an ORC until S phase starts then
cyclin is degraded, inactivating cdc2 enzyme. When bound and active
it prevents more than one initiation. At some point. a kinase
(Cdc6) coded by gene cdc6 either acts at the ORC or binds to it
directly (in budding yeast, in fission yeast, equivalent is cdc18).
TOPOISOMERASES:
Topos are either class I or II and all, including procaryotic, are members of a family. Proc topoII is usually a tetramer = A2B2 coded by gyrA and gyrB. Euc topoII is a dimer with the subunits C & N termini having homology with gyrA & gyrB respectively.
Responsible for chromatin condensation, chromosome segregation
(topoIIa
also found in centromer).
Yeast topoIIs have a heart shaped dimeric structure with each
half having a set of "jaws".
Mammals have 2 isozymes called topoIIa & b , a = 170kd from gene on chromosome 17, b = 180kd from chromosome
3 and is nucleolar. 70% homology between C-terminal domains. Human
TopoI = 67 kd. There are also "reverse" topoIs.
TopoI in plants is 90 kd coded by a 2370 bp orf. 40% homology with yeast topoI.
Mechanism is by scission of duplex & passing 2nd duplex through
the opening and ligating. Requires a configuration change of 35-40A,
ATPase activity and a transesterification between topo tyrosine
of each subunit and phosphodiester of backbone. Causes scission
with a 4 base stager, i.e., sticky ends.
TELOMERES AND TELOMERASES:
Apparently the eukaryotic HE can not synthesize
all the way to the very end of the DNA template and that Eukaryotic
chromosomes are not closed loops, each round of replicative synthesis
would shorten the chromosome. To prevent needed genes from being
"eaten away", there are extra sequences placed on the
ends of chromosomes called telomeres.
These are usually repetitive sequences:
human = 2-15kb of (TTAGGG)n
plants = (TTTAGGG)n
fission yeast = a 25bp sequence x 4
budding yeast = about 300 bp
Normal cells shorten the telomere each cell cycle --> Hayflick
limit
Regenerative cells like malignant, germ, and yeast possess a system
to regenerate the telomere using a telomerase.
Yeast have telomeres about 300 bp long that bind about 15 molecules
of a protein called Rap1. There seems to be a mechanism to count
these protein units. This mechanism may involve 2 accessory proteins
called Rap1 interacting factors (Rifs, Rif1 &Rif2). Mutation
studies show that mutant Rap1 causes run-away telomere lengthening,
mutated Rif1, moderate lengthening and mutated Rif2 excessive
lengthening. When about 15 Rap1s are bound, telomerase is inhibited.
Telomerase is a ribonucleoprotein terminal transferase which is
a fancy way to say that it is a reverse transcriptase. It has
it's own RNA template called htr bound as part of a complex.
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