Dasar-dasar Genetika

Agus Cahyono
Departemen Biokimia
Fakultas Kedokteran Universitas Surabaya

1
2
  Beadle and Tatum concluded that a gene is a segment of
genetic material that determines or codes for one enzyme:
the one gene–one enzyme hypothesis.
 Later this concept was broadened to one gene–one
polypeptide, because many genes code for proteins that
are not enzymes or for one polypeptide of a multisubunit
protein

3
 Colinearity of the coding nucleotide sequences of
DNA and mRNA and the amino acid sequence of a
polypeptide chain

4
 DNA: bahan genetik
Struktur kovalen dari asam nukleat
A. Asam nukleat adalah polimer dari nukleosida monofosfat
(nukleotida)
1. Tiap unit monomer terdiri atas:
 suatu basa nitrogen berupa purin atau pirimidin
 suatu gula pentosa berupa ribosa atau deoksiribosa
dalam bentuk (cincin ) furanosa
 suatu gugus fosfat yg teresterifikasi ke gula

2. Ikatan gula dengan basa membentuk nukleosida yg akan

membentuk nukleotida bila mengalami fosforilasi

5
 B. Nukleotida dapat terikat satu sama lain dengan ikatan
fosfodiester yg terbentuk antara gugus hidroksil -3’ dari
gula suatu nukleotida dengan fosfat -5’ dari gula nukleotida
lain

6
 Tatanama asam nukleat
A. polinukleotida
 pada ujung 5’ ada gugus fosfat yg disepakati selalu
ditempatkan pada sebelah kiri
 nukleotida-5’ adalah guanosin fosfat
 di ujung 3’ tidak terdapat gugus fosfat

 Bila pada nukleotida dilakukan perubahan kedudukan 2’

dari setiap gula, melalui penggantian gugus hidroksil
dengan H maka terbentuklah deoksiribosa  DNA

7
8
 B. poliribonukleotida
 Mengandung ribosa
 RNA
 Basa: guanin, sitosin, adenin, urasil

C. struktur DNA dan RNA

 1. DNA punya 2 deoksiribosa sebagai bagian gulanya
 2. DNA punya satu basa yg berbeda timin (5-metil uracil)
 3. RNA punya satu basa yg berbeda uracil

9
10
 DNA sebagai materi genetik
A. gen eukariot
 Pengemban materi genetik adalah DNA dalam seluruh
organisme kecuali pada virus tertentu (virus mozaik
tembakau, polio, influenza)

B. bukti untuk DNA sebagai bahan genetik

1. penelitian terdahulu dengan pneumokokus
 Griffith (1928)  mutan nirpatogen dari pneumokokus
dapat berubah menjadi bentuk patogen
 Avery, mcLeod, McCarthy (1944)  DNA sebagai bahan
genetik
11
 2. penyelidikan dengan bakteriofag T2
 A. Herriot (1951)  bakteriofag menyuntikkan asam
nukleat ke dalam sel, sedangkan lapisan protein ditinggal di
luar
 B. Hersley & Chase (1952)  menandai asam nukleat
bakteriofag T2 dengan fosfor radioaktif dan protein dengan
sulfur radioaktif. Setelah bakteriofag menginfeksi E. Coli,
fosfor radioaktif ada di dalam sel.

12
 Ciri khas struktur DNA
A. Struktur dasar DNA (Watson& Crick) heliks ganda:
 Dua rantai polideoksiribonukleotida yg berjalan antiparalel
 Tumpukan cincin hidrofobik terjadi bila bidang2 basa
berada tegak lurus terhadap sumbu heliks
 Ikatan hidrogen antar pasangan basa: A-T, G-C, hukum
Chargaf  jumlah A=T, G=C

13
 Complementarity between the two strands of
DNA

14
  Diameter heliks adalah 20u:
 Dalam kristal DNA basa-basa tersebut terpisah dengan
jarak 3,4A sepanjang sumbu heliks dan setiap basa
berputar dalam kedudukan 36* terhadap basa sebelumnya
(struktur heliks berulang setiap 10 pasangan basa)
 Dalam larutan, perulangan struktur DNA terjadi setiap 11
basa

15
  Dalam keadaan tertentu DNA berbentuk heliks putar kiri
(DNA Z)
 Informasi genetik disimpan sebagai urutan basa sepanjang
rantai

16
 B kesetaraan jumlah A-T, G-C
 Jumlah salah satu dari keempat basa standar dalam DNA
menentukan kandungan masing2 dari ketiga basa lainnya
 Susunan basa dapat ditetapkan dengan cara: sedimentasi
sampai mencapai suatu keseimbangan dalam larutan
cesium klorida dan pengukuran titik leleh
 Kandungan GC (fraksi mol) sama untuk DNA semua sel
dalam semua spesies tetapi bervariasi luas dari satu spesies
ke spesies lain

17
 C Perubahan basa
 Dalam DNA hewan dan tumbuh-tumbuhan 10% dari residu
C termetilasi melindungi DNA dari pemecahan nuklease
intrasel

18
 D. Ukuran molekul DNA
 Kromosom yg sangat sederhana seperti dalam virus simian
terdiri dari 5 atau 6 gen (5243 pasang basa) dan panjangnya
1,7um
 BM kromosom manusia 160x109 dalton mengandung
240x106 pasang basa dengan panjang 8,2 cm
 Panjang berbagai molekul DNA berbeda dalam kromosom
berbagai sel
 Molekul DNA berjalan sepanjang kromosom tanpa
berselang di sentromer

19
 E. Bentuk molekul DNA
 DNA kromosom mungkin lurus atau melingkar
 DNA membentuk kompleks dengan histon
 Bentuk supercoil DNA esensial untuk tahap replikasi,
transkripsi dan rekombinasi

20
21
 Pengguraian dan penggabungan rantai
heliks ganda DNA
A denaturasi
 peningkatan pH menyebabkan cincin nitrogen dari guanin
dan timin mengalami deprotonasi, penurunan pH
menyebabkan cincin nitrogen dari adenin, guanin dan
sitosin mengalami protonasi
 peningkatan keasaman dapat menyebabkan putusnya ikatan
glikosida/purin dan pada suhu yg tinggi menyebabkan
ikatan fosfodiester. Penggunaan alkali merupakan cara
terpilih untuk denaturasi DNA

22
  Kenaikan suhu dapat mengakibatkan denaturasi rantai
heliks ganda DNA
 Tm adalah suhu ketika 50% heliks ganda berpisah
 peleburan DNA dapat dipelajari dengan mengukur
serapan cahaya pada panjang gelombang 260nm,
peleburan menyebabkan pasangan basa kurang padat,
menghasilkan peningkatan penyerapan cahaya (efek
hiperkromik)

23
  Tm dipengaruhi sangat kuat oleh komposisi basa DNA.
1. DNA yg kaya pasangan GC Tm lebih tinggi dp AT
2. Tm dari DNA yang diambil dari spesies yg berlainan
dan diukur pada pH 7 dalam larutan garam isotonik
bervariasi garis lurus dengan kandungan GC dengan poli
AT sintetik yg mempunyai kira2 Tm 65 ͦC dan poli GC
sintetik yg mempunyai Tm 105 ͦC
3. DNA mamalia yg mengandung pasangan GC kira2 40%
mempunyai Tm sekitar 87 ͦC

24
 B. renaturasi
 Bila suhu peleburan DNA dupleks dengan cepat
diturunkan, struktur heliks ganda asli tidak terbentuk
kembali
 Namun, bila suhu ditahan pada sekitar 20-25 C di bawah
Tm struktur heliks ganda yg asli terbentuk kembali.
 Laju pembentukan kembali struktur berhubungan dengan
konsentrasi dari urutan2 komplementer

25
 Kromosom eukariot
A. sel eukariot
 Garis tengah 20u, mempunyai membran inti yg jelas dan
kromosom lebih dari 1, DNA hampir seluruhnya berada
dalam inti, sebagian kecil ditemukan di mitokondria atau
kloroplas
 Sel prokariot seperti E. Coli berbentuk silinder, berukuran
panjang 2u, garis tengah 1u, kromosom tunggal, tidak ada
batas yg jelas yg memisahkan kromosom dari sitoplasma

26
 B. DNA eukariot
1. DNA dikemas dalam kromosom, lalat buah ada 4
kromosom dan manusia 46
A. sel somatik mempunyai kromosom yg berpasangan
(homolog), masing2 berasal dari tiap induk  diploidi
B. setiap kromosom dalam fase metafase dari mitosis
mengandung 2 molekul DNA yg lengkap dan identik
(jadi ada 4 untai) karena DNA telah mengalami
replikasi selama fase S terdahuu dari daur sel

27
28
 2. sel diploid manusia mengandung kira2 6,4x109 pasangan
basa DNA, 1000x lebih banyak daripada E.coli
A. basa ini tersebar di 46 kromosom (masing2
mengandung 1,5x108 pasangan basa)
B. bila direntang, 1,5x108 pasangan basa tersebut akan
berukuran sepanjang 47mm

29
 C. pembagian kronologis dari daur sel eukariot
1. pada tahap metafase dari mitosis kromosom dapat
dilihat dengan mikroskop cahaya sebagai kesatuan
tersendiri
2. metafase disusul oleh tahap G1
3. sintesis, S  replikasi DNA
4. G2, fase antara fase S dan metafase berikutnya

30
 D. Kromatin (gabungan DNA dengan protein)
1. Histon
 DNA eukariot bergabung dengan protein basa yg bernama
histon dan dengan protein lain ygt disebut protein
kromosom
 Histon adalah protein kecil yg bermuatan positif
 Histon ada 5 kelas (H1, H2A, H2B, H3, H4) yg berat
molekulnya bervariasi 11,3 kdal-21 kdal
 Protein basa ini mempunyai urutan asam amino yg
dipertahankan selama 1 eon (1,2x109 tahun)

31
32
 2. nukleosom  penyusun kromatin
A. nukleosom mempunyai 1 pusat yg terdiri atas suatu
oktamer histon yg mengikat secara nonkovalen 140
pasangan basa DNA yg menyelubungi histon dalam
bentuk superheliks ke kiri
B. sisa pasangan basa kira2 60 membentuk rantai yg
menghubungkan merjan-merjan tersebut satu sama lain
C. suatu molekul H1 tunggal mungkin ada di sebelah luar
pusat, dekat DNA linker
D. nukleosom merupakan pemampatan DNA tahap 1

33
34
 3. Eukromatin dan heterokromatin

35
 E. DNA sitoplasma dalam organel sitoplasma
1. Baik DNA mitokondria maupun kloroplas tidak disertai
histon
2. DNA mitokondria sel binatang adalah: untai ganda,
melingkar, mempunyai panjang kira2 15.000 pasangan
basa
3. urutan DNA mitokondria mengkode hanya utk 5% saja
dari unsur protein dari struktur dan fungsi mitokondria

36
 Heterogenitas dari DNA eukariot
A. urutan unik (gen dengan salinan tunggal)
1. kira2 70% dari DNA kebanyakan eukariot terdiri atas
urutan unik yg renaturasi sangat lambat
2. urutan yg mengandung sandi (ekson) dipisahkan oleh
urutan penyelang (intron)

B. urutan dengan pengulangan sangat tinggi

1. urutan yg sangat berulang ini dikenal sebagai DNA
satelit, panjang antara 2-10 pasangan basa dan berulang
secara berurutan 105- 107 kali dalam genom

37
 A. urutan ini mencapai 15% dari genom
B. tidak disalin
C. terpusat di daerah sentromer dari kromosom

2. urutan yg sangat berulang ini renaturasi sangat cepat

38
 C. urutan dengan pengulangan sedang
 Beberapa produk gen disandikan oleh urutan DNA yg
berulang berurutan (in tandem), kerap kali lebih dari 100
kali, misalnya
1. rRNA
2. tRNA
3. gen histon
4. urutan DNA yg tidak disalin

39
 D. Urutan berulang terbalik mencapai 6% dari genom
manusia, akan tetapi fungsinya tidak diketahui

40
41
42
43
 Genetic Mutation

Agus Cahyono
Departemen Biokimia
Fakultas Kedokteran Universitas Surabaya

44
 Genetic mutation

45
 DNA Repair
 Damaged proteins & RNA molecules  quickly replaced
by using information encoded in the DNA, but DNA
molecules themselves are irreplaceable.
 Maintaining the integrity of the information in DNA is a
cellular imperative, supported by an elaborate set of DNA
repair systems.
 DNA can become damaged by a variety of processes, some
spontaneous, others catalyzed by environmental agents.
 Replication itself can very occasionally damage the
information content in DNA when errors introduce
mismatched base pairs (such as G paired with T)

46
 Mutations Are Linked to Cancer
 The best way to illustrate the importance of DNA repair is
to consider the effects of unrepaired DNA damage (a
lesion).
 The most serious outcome is a change in the base sequence
of the DNA, which, if replicated and transmitted to future
cell generations, becomes permanent.
 A permanent change in the nucleotide sequence of DNA is
called a mutation.

47
  Mutations can involve the replacement of one base pair
with another (substitution mutation) or the addition or
deletion of one or more base pairs (insertion or deletion
mutations).
 If the mutation affects nonessential DNA or if it has a
negligible effect on the function of a gene, it is known as a
silent mutation.
 Rarely, a mutation confers some biological advantage.
 Most nonsilent mutations, however, are deleterious

48
 Type of mutations
Substitution
 mutation that exchanges one base for another (i.e., a change
in a single "chemical letter" such as switching an A to a G).
 such a substitution could:change a codon to one that
encodes a different amino acid and cause a small change in
the protein produced. For example,sickle cell anemia is
caused by a substitution in the beta-hemoglobin gene,
which alters a single amino acid in the protein produced.
 change a codon to one that encodes the same amino acid
and causes no change in the protein produced (silent
mutations)

49
  change an amino-acid-coding codon to a single "stop"
codon and cause an incomplete protein  serious effects
since the incomplete protein probably won't function.

50
  Insertion
mutations in which extra base pairs are inserted into a new
place in the DNA.

51
  Deletion
mutations in which a section of DNA is lost, or deleted.

52
  Frameshift
Since protein-coding DNA is divided into codons three
bases long, insertions & deletions can alter a gene  its
message is no longer correctly parsed (frameshifts).
 For example, consider the sentence, "The fat cat sat." Each
word represents a codon. If we delete the first letter and
parse the sentence in the same way, it doesn't make sense.
 In frameshifts, a similar error occurs at the DNA level,
causing the codons to be parsed incorrectly. This usually
generates truncated proteins that are as useless as "hef atc
ats at" is uninformative.

53
54
 The causes of mutations
1. DNA fails to copy accurately
Most of the mutations that we think matter to evolution are
"naturally-occurring." For example, when a cell divides, it
makes a copy of its DNA — and sometimes the copy is not
quite perfect. That small difference from the original DNA
sequence is a mutation.

55
 2. External influences can create mutations
 Mutations can also be caused by exposure to specific
chemicals or radiation.
 These agents cause the DNA to break down. This is not
necessarily unnatural — even in the most isolated and
pristine environments, DNA breaks down.
 Nevertheless, when the cell repairs the DNA, it might not
do a perfect job of the repair. So the cell would end up with
DNA slightly different than the original DNA and hence, a
mutation.

56
 The effects of mutations
 Since all cells in our body contain DNA, there are lots of
places for mutations to occur; however, some mutations
cannot be passed on to offspring and do not matter for
evolution.
 Somatic mutations occur in non-reproductive cells and
won't be passed onto offspring
 The only mutations that matter to large-scale evolution are
those that can be passed on to offspring. These occur in
reproductive cells like eggs and sperm and are called germ
line mutations.

57
 Effects of germ line mutations
 No change occurs in phenotype.
Some mutations don't have any noticeable effect on the
phenotype of an organism. This can happen in many
situations: perhaps the mutation occurs in a stretch of DNA
with no function, or perhaps the mutation occurs in a
protein-coding region, but ends up not affecting the amino
acid sequence of the protein.
 Small change occurs in phenotype.
A single mutation caused this cat's ears to curl backwards
slightly.

58
59
  Big change occurs in phenotype.
Some really important phenotypic changes, like DDT
resistance in insects are sometimes caused by single
mutations. A single mutation can also have strong negative
effects for the organism. Mutations that cause the death of
an organism are called lethals — and it doesn't get more
negative than that.

60
 Little mutations with big effects:
Mutations to control genes
 Mutations are often the victims of bad press — unfairly
stereotyped as unimportant or as a cause of genetic disease.
While many mutations do indeed have small or negative
effects, another sort of mutation gets less airtime.
Mutations to control genes can have major (and sometimes
positive) effects.

61
  Some regions of DNA control other genes, determining
when and where other genes are turned "on".
 Mutations in these parts of the genome can substantially
change the way the organism is built. The difference
between a mutation to a control gene and a mutation to a
less powerful gene is a bit like the difference between
whispering an instruction to the trumpet player in an
orchestra versus whispering it to the orchestra's conductor.

62
63
  Many organisms have powerful control genes that
determine how the body is laid out.
 For example, Hox genes are found in many animals
(including flies and humans) & designate where the head
goes and which regions of the body grow appendages.
 Such master control genes help direct the building of body
"units," such as segments, limbs, and eyes. So evolving a
major change in basic body layout may not be so unlikely;
it may simply require a change in a Hox gene and the favor
of natural selection.

64
  Mutations to control genes can transform one body part
into another. Scientists have studied flies
carrying Hox mutations that sprout legs on their foreheads
instead of antennae!

65
 A case study of the effects of mutation:
Sickle cell anemia
 Sickle cell anemia is a genetic disease with severe
symptoms, including pain and anemia.
 The disease is caused by a mutated version of the gene that
helps make hemoglobin — a protein that carries oxygen in
red blood cells.
 People with two copies of the sickle cell gene have the
disease. People who carry only one copy of the sickle cell
gene do not have the disease, but may pass the gene on to
their children.

66
  The mutations that cause sickle cell anemia have been
extensively studied and demonstrate how the effects of
mutations can be traced from the DNA level up to the level
of the whole organism.
 Consider someone carrying only one copy of the gene. She
does not have the disease, but the gene that she carries still
affects her, her cells, and her proteins:

67
 1. There are effects at the DNA level

68
 2. There are effects at the protein level
 Normal hemoglobin (left) and hemoglobin in sickled red
blood cells (right) look different; the mutation in the DNA
slightly changes the shape of the hemoglobin molecule,
allowing it to clump together.

69
 3. There are effects at the cellular level
When red blood cells carrying mutant hemoglobin are
deprived of oxygen, they become "sickle-shaped" instead
of the usual round shape (see picture). This shape can
sometimes interrupt blood flow.

70
 4. There are negative effects at the whole organism level
Under conditions such as high elevation and intense
exercise, a carrier of the sickle cell allele may occasionally
show symptoms such as pain and fatigue.

71
 5. There are positive effects at the whole organism level
Carriers of the sickle cell allele are resistant to malaria,
because the parasites that cause this disease are killed
inside sickle-shaped blood cells.

72
 Mutations are random
 Mutations can be beneficial, neutral, or harmful for the
organism, but mutations do not "try" to supply what the
organism "needs."
 Factors in the environment may influence the rate of
mutation but are not generally thought to influence the
direction of mutation.
 For example, exposure to harmful chemicals may increase
the mutation rate, but will not cause more mutations that
make the organism resistant to those chemicals. In this
respect, mutations are random — whether a particular
mutation happens or not is unrelated to how useful that
mutation would be.

73
  For example, in the U.S. where people have access to
shampoos with chemicals that kill lice, we have a lot of lice
that are resistant to those chemicals. There are two possible
explanations for this:
 Hipotesis A Hipotesis B

Resistant strains of lice were always there — Exposure to lice shampoo actually
and are just more frequent now because all caused mutations for resistance to
74 the non-resistant lice died a sudsy death the shampoo.
  Scientists generally think that the first explanation is the
right one and that directed mutations, the second possible
explanation relying on non-random mutation, is not correct.
 Researchers have performed many experiments in this area.
Though results can be interpreted in several ways, none
unambiguously support directed mutation. Nevertheless,
scientists are still doing research that provides evidence
relevant to this issue.

75
  In addition, experiments have made it clear that many
mutations are in fact random, and did not occur because the
organism was placed in a situation where the mutation
would be useful.
 For example, if you expose bacteria to an antibiotic, you
will likely observe an increased prevalence of antibiotic
resistance. Esther and Joshua Lederberg determined that
many of these mutations for antibiotic resistance existed in
the population even before the population was exposed to
the antibiotic — and that exposure to the antibiotic did not
cause those new resistant mutants to appear.

76
 The Lederberg experiment
 In 1952, Esther and Joshua Lederberg performed an
experiment that helped show that many mutations are
random, not directed.
 In this experiment, they capitalized on the ease with which
bacteria can be grown and maintained.
 Bacteria grow into isolated colonies on plates. These
colonies can be reproduced from an original plate to new
plates by "stamping" the original plate with a cloth and then
stamping empty plates with the same cloth. Bacteria from
each colony are picked up on the cloth and then deposited
on the new plates by the cloth.

77
  Esther and Joshua hypothesized that antibiotic resistant
strains of bacteria surviving an application of antibiotics
had the resistance before their exposure to the antibiotics,
not as a result of the exposure. Their experimental set-up is
summarized below:

78
79
80
 DNA repair
 The genome of a typical mammalian cell accumulates
many thousands of lesions during a 24-hour period.
 However, as a result of DNA repair, fewer than 1 in 1,000
becomes a mutation.
 DNA is a relatively stable molecule, but in the absence of
repair systems, the cumulative effect of many infrequent
but damaging reactions would make life impossible

81
82
 All Cells Have Multiple DNA Repair Systems
 The number and diversity of repair systems reflect both the
importance of DNA repair to cell survival and the diverse
sources of DNA damage (Table 25–5).
 Some common types of lesions, such as pyrimidine dimers,
can be repaired by several distinct systems.
 Many DNA repair processes also appear to be
extraordinarily inefficient energetically—an exception to
the pattern observed in the metabolic pathways, where
every ATP is generally accounted for and used optimally.
 When the integrity of the genetic information is at stake,
the amount of chemical energy invested in a repair process
seems almost irrelevant.

83
84
  DNA repair is possible largely because the DNA molecule
consists of two complementary strands.
 DNA damage in one strand can be removed and accurately
replaced by using the undamaged complementary strand as
a template.
 We consider here the principal types of repair systems,
beginning with those that repair the rare nucleotide
mismatches that are left behind by replication.

85
 Mismatch Repair
 Correction of the rare mismatches left after replication in E.
coli improves the overall fidelity of replication by an
additional factor of 102 to 103.
 The mismatches are nearly always corrected to reflect the
information in the old (template) strand, so the repair
system must somehow discriminate between the template
and the newly synthesized strand.
 The cell accomplishes this by tagging the template DNA
with methyl groups to distinguish it from newly
synthesized strands.
 The mismatch repair system of E. coli includes at least 12
protein components (Table 25–5) that function either in
strand discrimination or in the repair process itself.

86
 FIGURE 25–20 Methylation and mismatch repair.
Methylation of DNA strands can serve to distinguish
parent (template) strands from newly synthesized strands in
E. coli DNA, a function that is critical to mismatch repair (see
Fig. 25–21). The methylation occurs at the N6 of adenines in
(5)GATC sequences. This sequence is a palindrome (see Fig.
87 8–20), present in opposite orientations on the two strands
 FIGURE 25–21 A model for the early steps of methyl-directed mismatch repair. The proteins
involved in this process in E. coli have been purified (see Table 25–5). Recognition of the sequence
(5)GATC and of the mismatch are specialized functions of the MutH and MutS proteins, respectively. The
MutL protein forms a complex with MutS at the mismatch. DNA is threaded through this complex such that
the complex moves simultaneously in both directions along the DNA until it encounters a MutH protein
bound at a hemimethylated GATC sequence. MutH cleaves the unmethylated strand on the 5 side of the G in
this sequence. A complex consisting of DNA helicase II and one of several exonucleases then degrades the
88
unmethylated DNA strand from that point toward the mismatch
89
 Base-Excision Repair
 Every cell has a class of enzymes called DNA glycosylases
that recognize particularly common DNA lesions (such as
the products of cytosine and adenine deamination; see Fig.
8–33a) and remove the affected base by cleaving the N-
glycosyl bond.
 This cleavage creates an apurinic or apyrimidinic site in the
DNA, commonly referred to as an AP site or abasic site.
Each DNA glycosylase is generally specific for one type of
lesion

90
 FIGURE 25–23 DNA repair by the base-excision repair pathway. 1 A DNA glycosylase
recognizes a damaged base and cleaves between the base and deoxyribose in the backbone. 2 An AP
endonuclease cleaves the phosphodiester backbone near the AP site. 3 DNA polymerase I initiates
repair synthesis from the free 3 hydroxyl at the nick, removing (with its 5n3 exonuclease activity) a
portion of the damaged strand and replacing it with undamaged DNA. 4 The nick remaining after
91
DNA polymerase I has dissociated is sealed by DNA ligase.
 Nucleotide-Excision Repair
 DNA lesions that cause large distortions in the helical
structure of DNA generally are repaired by the nucleotide-
excision system, a repair pathway critical to the survival of
all free-living organisms.
 In nucleotide-excision repair (Fig. 25–24), a multisubunit
enzyme hydrolyzes two phosphodiester bonds, one on
either side of the distortion caused by the lesion.
 In E. coli and other prokaryotes, the enzyme system
hydrolyzes the fifth phosphodiester bond on the 3 side and
the eighth phosphodiester bond on the 5 side to generate a
fragment of 12 to 13 nucleotides (depending on whether the
lesion involves one or two bases).

92
  In humans and other eukaryotes, the enzyme system
hydrolyzes the sixth phosphodiester bond on the 3 side and
the twenty-second phosphodiester bond on the 5 side,
producing a fragment of 27 to 29 nucleotides.
 Following the dual incision, the excised oligonucleotides
are released from the duplex and the resulting gap is
filled—by DNA polymerase I in E. coli and DNA
polymerase in humans.
 DNA ligase seals the nick.

93
 FIGURE 25–24 Nucleotide-excision repair in E. coli and humans.The general pathway of nucleotide-
excision repair is similar in all organisms. 1 An excinuclease binds to DNA at the site of a bulky lesion and cleaves
the damaged DNA strand on either side of the lesion. 2 The DNA segment—of 13 nucleotides (13 mer) or 29
94 nucleotides (29 mer)—is removed with the aid of a helicase. 3 The gap is filled in by DNA polymerase, and 4 the
remaining nick is sealed with DNA ligase.
 Direct Repair
 Several types of damage are repaired without removing a
base or nucleotide.
 The best-characterized example is direct photoreactivation
of cyclobutane pyrimidine dimers, a reaction promoted by
DNA photolyases.
 Pyrimidine dimers result from an ultraviolet light–
induced reaction, and photolyases use energy derived from
absorbed light to reverse the damage (Fig. 25–25).

95
  Photolyases generally contain two cofactors that serve as
light-absorbing agents, or chromophores.
 One of the chromophores is always FADH.
 In E. coli and yeast, the other chromophore is a folate.
 The reaction mechanism entails the generation of free
radicals.
 DNA photolyases are not present in the cells of placental
mammals (which include humans).

96
97
98
99
100
  Lehninger
 Evolution.berkeley.edu

101