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Mutations in the SCN5A gene can also contribute to

idiopathic ventricular ﬁbrillation (IVF), a common cause

of sudden cardiac death. A variant SCN5A gene

segregating with IVF in an affected family contained

two missense mutations, affecting amino acid residues in

the extracellular loops between transmembrane seg-

ments S1 and S2 of domain III and between S3 and S4 of

domain IV. The latter extracellular loop is involved in

the coupling between channel activation and fast

inactivation. The mutations affect the voltage depen-

dence of steady-state inactivation of the sodium

channels, shifting it more than 10 mV towards more

positive potentials and accelerating rates of recovery

from inactivation. Frameshift mutations and splice-site

mutations in SCN5A have also been shown to be

associated with IVF in affected families.

Mutations of the mouse Scn8a gene of the mouse

have been shown to cause motor endplate disease, a

lethal skeletal muscle atrophy. Less severe phenotypes

involving ‘jolting’, a rhythmic tremor of the head and

body and unsteady gait, are associated with missense

mutations in the Scn8a gene.

A mutation affecting the sodium channel

1-subunit

has been shown to be the cause of inherited febrile

seizures. The mutation alters a cysteine residue involved

in stabilizing the immunoglobulin-like fold in the

extracellular domain of the

1-subunit that is involved

in modulating sodium channel gating.

Toxins and Channel Modiﬁers

The voltage-gated sodium channels are subject to

modiﬁcation by a wide range of toxins produced by

animals, for use in predation or defence, or by plants

as protection against herbivores. Six distinguishable

toxin-binding sites have been identiﬁed on sodium

channels, as summarized in Table II. The groups of

toxins have various effects on sodium channels, from

complete block to prolongation of activation.

The guanidinium-containing compounds, tetrodo-

toxin, isolated from the internal organs of the puffer ﬁsh

and its relatives in the family Tetraodontidae,and

saxitoxin, from marine dinoﬂagellates, act as selective,

reversible blockers at nanomolar concentrations. These

membrane impermeant toxins bind at the extracellular

opening of the channel pore and cause prolonged block of

nerve and muscle action potentials. Sodium channels in

heart and sensory neurons of dorsal root ganglia are

relatively insensitive to tetrodotoxin and saxitoxin. The

peptide

-conotoxins, isolated from the venom of the ﬁsh-

hunting cone snails of the Conus genus, also block muscle

action potentials by binding to the extracellular opening

of the pore of the skeletal muscle sodium channel. The

venom, which is injected via a harpoon-like disposable

tooth, rapidly results in paralysis and death of the prey.

A group of lipid-soluble alkaloids, including batra-

chotoxin, aconitine, veratridine, and grayanotoxin I,

cause hyperexcitability and cardiac arrhythmias by

promoting prolonged opening of sodium channels and

long-lasting membrane depolarization. Batrachotoxin,

secreted by the skin of Columbian frogs of the genus

Phyllobates, is used by indigenous natives to make poison

arrows for hunting. Brevetoxin, a fused cyclic ether

produced by the marine dinoﬂagellate, Ptychodiscus

brevis, the organism responsible for ‘red tides’, acts like

the alkaloids, but via a different binding site on the

sodium channel.

The peptide toxins of the scorpion

-toxin family,

produced by north African genera Androctinus, Buthus,

and Leirus and the American genus Centruroides, act to

TABLE I

Genes Encoding Mammalian Sodium Channel

-Subunits

Gene Location Channel type Tissue speciﬁcity Associated disease

SCN1A 2q24 Na

1.1 CNS, PNS Epilepsy

SCN2A 2q23–24 Na

1.2 CNS Neonatal-infantile seizures

SCN3A 2q24 Na

1.3 CNS

SCN4A 17q23–25 Na

1.4 Sk.m. Myotonia (HYPP, PMC)

SCN5A 3p21 Na

1.5 Heart, Uninnervated sk.m. Long QT syndrome

SCN8A 12q13 Na

1.6 CNS,PNS Cerebellar ataxia

SCN9A 2q24 Na

1.7 Schwann cells

SCN10A 3p22–24 Na

1.8 DRG

SCN11A 3p21–24 Na

1.9 PNS

SCN6A

2q21–23 Na

Heart, uterus, sk.m.

This condition is associated with mutations in the mouse SCN8A gene.

This gene was originally designated SCN6A in human and SCN7A in mouse. The two genes represent homologues and the

SCN6A symbol will probably be deleted. CNS ¼ central nervous system; PNS ¼ peripheral nervous system; sk.m. ¼ skeletal

muscle; DRG ¼ dorsal root ganglion neurons.

VOLTAGE-SENSITIVE NA

CHANNELS 413

prolong the action potential duration in muscle and

nerves by inhibiting sodium channel inactivation. The

nonhomologous sea anemone toxins act at the same site

and produce similar effects.

A second group of peptide-based scorpion toxins, the

-toxins, slow both activation and inactivation of

sodium channels in skeletal muscle. The consequence

of this modulation is that channels close very slowly at

rest. The resulting inward Na

currents support

repetitive ﬁring in response to minimal stimulation,

producing hyperexcitability, with heavy perspiration

and muscle tremor.

The pyrethrins, natural insecticides produced by

ﬂowers of the genus Chrysanthemum, and their syn-

thetic analogues the pyrethroids, are organic esters that

prolong sodium channel activation and inhibit inacti-

vation in nerve axons. The consequence of this effect is

repetitive ﬁring and membrane depolarization,

leading to lethal paralysis in insects. The synthetic

insecticide dichlorodiphenyltrichloroethane (DDT) acts

in a similar fashion.

Several local anesthetics, such as lidocaine and

procaine, are lipid-soluble tertiary amines that block

sodium channels in nerve axons to inhibit propaga-

tion of action potentials. Various sodium channel

blockers, including local anesthetics and alkaloids, are

being used clinically as neuroprotective agents in

ischemic stroke patients and as antiarrhythmic agents

in heart disease.

FURTHER READING

Aidley, D. J., and Stanﬁeld, P. R. (1996). Ion Channels: Molecules in

Action. Cambridge University Press, Cambridge, UK.

Catterall, W. A. (2000). From ionic currents to molecular mechanisms:

The structure and function of voltage-gated sodium channels.

Neuron 26, 13–25.

Goldin, A. L., Barchi, R. L., Caldwell, J. H., Hofmann, F., Howe, J. R.,

Hunter, J. C., Kallen, R. G., Mandel, G., Meisler, M. H.,

Netter, Y. B., Noda, M., Tamkun, M. M., Waxman, S. G., Wood,

J. N., and Catterall, W. A. (2000). Nomenclature of voltage-gated

sodium channels. Neuron 28, 365–368.

Hille, B. (2001). Ion Channels of Excitable Membranes. 3rd edition,

Sinauer Associates, Sunderland, Massachusetts.

BIOGRAPHY

William J. Brammar is a Professor of Biochemistry and Pro-Vice-

Chancellor (Research) at the University of Leicester. He has a long-

term interest in the regulation of expression of both prokaryotic

and eukaryotic genes. He holds a Ph.D. from University College,

London and was a postdoctoral fellow at Stanford University. He

has written texts on molecular cloning and ion channels and is a

member of the European Molecular Biology Organization.

VOLTAGE-SENSITIVE NA

CHANNELS 415

Von Hippel-Lindau (VHL) Protein

Ronald C. Conaway and Joan Weliky Conaway

Stowers Institute for Medical Research, Kansas City, Missouri, USA

The Von Hippel-Lindau (VHL) protein is a tumor suppressor.

Mutations in the VHL protein can give rise to tumors of

multiple organ systems, including the central nervous system,

the endocrine system, and the kidney. The VHL protein

functions as a subunit of a multiprotein ubiquitin ligase that

negatively regulates expression of a large collection of hypoxia-

inducible genes controlled by hypoxia-inducible transcription

factors (HIFs). The VHL ubiquitin ligase prevents inappropri-

ate expression of these hypoxia-inducible genes when cells are

grown in a plentiful supply of oxygen by targeting HIFs for

rapid ubiquitylation and degradation by the proteasome.

Clinical Consequences

of VHL Mutations

Mutations in the VHL gene are found in a variety of

human diseases, including VHL disease, sporadic clear

cell renal carcinoma, sporadic hemangioblastoma, and

congenital polycythemia. VHL disease is an autosomal

dominant familial cancer syndrome. In VHL kindreds,

inactivation of both copies of the VHL gene can give rise

to a variety of highly vascularized tumors, including

clear cell renal carcinoma, cerebellar hemangioblastoma

and hemangioma, retinal angioma, and pheochromo-

cytoma. VHL mutations are also responsible for the

majority of cases of sporadic clear cell renal carcinoma

and for sporadic hemangioblastoma. In addition, VHL

mutations are believed to give rise to some forms of

polycythemia, a disorder characterized by elevated

levels of expression of the hypoxia-inducible erythro-

poietin gene.

Function of the VHL Protein

The VHL protein is present in eukaryotes from worms to

mammals, where it performs an evolutionarily con-

served function as a subunit of a multiprotein ubiquitin

ligase that negatively regulates expression of a large

number of hypoxia-inducible genes controlled

by hypoxia-inducible transcription factors (HIFs).

The expression of these hypoxia-inducible genes is

repressed in normal cells grown in a plentiful supply of

oxygen (normoxic conditions), but is strongly induced in

cells starved for oxygen (hypoxic conditions). The VHL

ubiquitin ligase prevents the inappropriate expression of

these hypoxia-inducible genes when cells are grown in

normoxic conditions by targeting HIFs for rapid

ubiquitylation and degradation by the proteasome.

When cells are starved for oxygen, ubiquitylation of

HIFs by the VHL ubiquitin ligase is blocked. Under these

hypoxic conditions, cellular HIF levels rise, and HIFs

enter the nucleus, bind to hypoxia response elements

(HREs) in the promoters of hypoxia-inducible genes,

and activate their transcription and expression.

The cellular levels of HIFs correlate well with the

levels of expression of hypoxia-inducible genes in both

normoxic and hypoxic conditions (Figure 1). In normal

cells growing under normoxic conditions, HIFs are

present in cells at low levels, and expression of hypoxic-

inducible genes is barely detectable. Under hypoxic

conditions, HIFs are present at relatively high levels, and

expression of hypoxia-inducible genes is robust. Under

both normoxic and hypoxic conditions in cells lacking a

functional VHL protein, HIFs are present in cells at high

levels, and maximal expression of hypoxia-inducible

genes is observed. Inappropriate overexpression of

hypoxia-inducible genes in cells lacking a functional

VHL protein may contribute to the pathology of VHL-

associated diseases. Among these are the genes encoding

vascular endothelial growth factor (VEGF), transform-

ing growth factor a (TGFa), and erythropoietin (EPO).

VEGF is a secreted protein that appears to play a major

role in the vascularization of VHL tumors by promoting

ingrowth into tumors of blood vessels needed to carry

oxygen and nutrients to the tumor cells. TGFa is also a

secreted protein that appears to function as an autocrine

growth factor for some types of VHL tumor cells and

may be responsible for promoting their uncontrolled

proliferation. An elevated EPO level in the serum of

polycythemic patients is a hallmark of the disease.

STRUCTURE OF THE VHL

BIQUITIN LIGASE

Puriﬁcation of the VHL ubiquitin ligase revealed that it is

composed of multiple proteins. In addition to the VHL

protein, the VHL ubiquitin ligase includes the elongin B

and C proteins, cullin protein family member Cul2, and

RING ﬁnger protein Rbx1 (Figure 2). In the VHL

ubiquitin ligase, the VHL protein functions to recruit

HIFs for ubiquitylation by interacting directly with

them. The elongin B and C proteins bind stably to each

other to form an adaptor that links the VHL protein to a

Cul2-Rbx1 heterodimeric module. The Cul2-Rbx1

module functions to recruit the E2 ubiquitin conjugating

enzyme Ubc5, which is responsible for ubiquitylation of

HIFs. The VHL ubiquitin ligase is the founding member

of a family of elongin BC-based ubiquitin ligases that

include Cul2-Rbx1 or Cul5-Rbx1 modules. In the VHL

ubiquitin ligase, the VHL protein binds directly to

elongin BC through a conserved elongin BC-binding

site motif (BC-box) with consensus sequence

[(A,P,S,T)LXXXCXXX(A,I,L,V)]. In other elongin

BC-based ubiquitin ligases, the VHL protein is replaced

by one of many known BC-box proteins, including

members of the family of more than thirty suppressors of

cytokine signaling (SOCS) proteins, which play import-

ant roles in a variety of signal transduction pathways.

REGULATION OF HIF UBIQUITYLATION

BY THE

VHL UBIQUITIN LIGASE

To date, three members of the HIF family of transcrip-

tion factors have been identiﬁed in mammalian cells and

shown to be regulated by the VHL ubiquitin ligase. HIFs

are heterodimeric DNA-binding transcription factors

that bind to HREs in the promoters of hypoxia-inducible

genes and activate their expression. In mammalian cells,

HIF heterodimers are composed of one of three related

HIFa proteins and a common subunit, the aryl

hydrocarbon receptor nuclear translocator protein

(ARNT). The HIFa and ARNT proteins are basic

helix-loop-helix (bHLH) transcription factors that all

contain an additional conserved domain, referred to as

the PAS domain (PER-ARNT-SIM domain), of unknown

function. The HIFa subunits, but not the ARNT

subunits, of HIFs are targets of the VHL ubiquitin

ligase. Whereas the ARNT subunit of HIFs is present in

normoxic and hypoxic cells at similarly high levels, the

HIFa subunits are continuously synthesized but rapidly

ubiquitylated by the VHL ubiquitin ligase and degraded

by the proteasome in normal cells grown under

normoxic conditions. In hypoxic cells, ubiquitylation

of HIFa subunits is attenuated. Under these conditions,

cellular HIFa levels rise, and HIFa subunits hetero-

dimerize with ARNT, enter the nucleus, and activate

expression of hypoxia-inducible genes.

HIFa ubiquitylation by the VHL ubiquitin ligase is

regulated by oxygen. Elegant studies have revealed that

binding of HIFa subunits to the VHL protein and their

recruitment to the VHL ubiquitin ligase depends on

hydroxylation of a speciﬁc HIFa proline in a HIFa

region referred to as the oxygen-dependent degradation

domain (ODD). The HIFa ODD is an approximately

20aminoacidHIFa region that is essential for

interaction of HIFa with the VHL protein and ubiqui-

tylation of HIFa by the VHL ubiquitin ligase. The ODD

is a portable domain capable of promoting oxygen-

dependent degradation of a variety of ODD-containing

fusion proteins. Binding of HIFa to the VHL protein

depends on hydroxylation of a proline in the ODD by an

iron- and oxoglutarate-dependent dioxygenase of the

EGL-9 protein family. The EGL-9 proline hydroxylase

uses O

as the oxygen donor for hydroxylation of HIFa

VHL (+) VHL (–)

Normoxia

Hypoxia

Normoxia

Expression of hypoxia-inducible genes

and abundance of HIFa protein

FIGURE 1 Regulation of expression of hypoxia-inducible genes and

HIFa protein levels in normoxia and hypoxia. VHL (þ), normal cells;

VHL (2 ), cells lacking a functional VHL protein.

FIGURE 2 Architecture of the VHL ubiquitin ligase. Ub, ubiquitin;

E2 (Ubc5), E2 ubiquitin conjugating enzyme Ubc5; EloB, elongin B;

EloC, elongin C.

VON HIPPEL-LINDAU (VHL) PROTEIN 417

subunits in a reaction with kinetics proportional to the

concentration of cellular O

. In cells grown in hypoxic

conditions, HIFa hydroxylation is signiﬁcantly reduced,

and the nonhydroxylated HIFa subunits are no longer

substrates for ubiquitylation by the VHL ubiquitin

ligase. Thus, oxygen-dependent hydroxylation of HIFa

serves as a convenient molecular switch for regulation of

expression of hypoxia-inducible genes.

FURTHER READING

Conaway, R. C., and Conaway, J. W. (2002). The Von Hippel-Lindau

tumor suppressor complex and regulation of hypoxia-inducible

transcription. Adv. Cancer Res. 85, 1– 12.

Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., Jr., and

Pavletich, N. P. (2002). Structure of an HIF-1alpha-pVHL

complex: Hydroxyproline recognition in signaling. Science 296,

1886–1889.

Pugh, C. W., and Ratcliffe, P. J. (2003). The Von Hippel-Lindau tumor

suppressor, hypoxia-inducible factor-1 (HIF-1) degradation, and

cancer pathogenesis. Semin. Cancer Biol. 13, 83–89.

Semenza, G. L. (2001). Hypoxia-inducible factor 1: Oxygen homeo-

stasis and disease pathophysiology. Trends Mol. Med. 8, 345–350.

Stebbins, C. E., Kaelin, W. G., and Pavletich, N. P. (1999). Structure of

the VHL-elongin C-elongin B complex: Implications for VHL

tumor suppressor function. Science 284, 455–461.

BIOGRAPHY

Ronald C. Conaway and Joan Weliky Conaway are Senior Scientists

at the Stowers Institute for Medical Research. Research in their

laboratory is aimed at contributing to an understanding of the

molecular mechanisms governing the synthesis of eukaryotic messen-

ger RNA. They hold Ph.D.s from the Stanford University School

of Medicine.

418 VON HIPPEL-LINDAU (VHL) PROTEIN

XPV DNA Polymerase and

Ultraviolet Damage Bypass

Alan R. Lehmann

University of Sussex, Brighton, UK

DNA damage blocks the progress of the replication fork.

In order to bypass the damage, cells have evolved special

DNA polymerases, which are uniquely able to replicate

damaged DNA. DNA polymerase

(eta) is the enzyme

that carries out this bypass function for the major form of

UV damage in DNA. A deﬁciency in this polymerase leads

to a severe human condition, the variant form of

xeroderma pigmentosum.

XP Variants

All cells sustain many kinds of DNA damage. This can

be generated either endogenously from hydrolysis and

oxidation reactions or exogenously from exposure to

carcinogens. One of the most prevalent DNA dama-

ging agents is UV radiation from the sun, which

generates photoproducts in the DNA of cells in

exposed areas of the skin. The most important

photodimer, namely the cyclobutane pyrimidine

dimer (CPD) and the pyrimidine 6-4 pyrimidone

photodimer (6-4 PP) are removed from cellular DNA

by the process of nucleotide excision repair (NER).

The importance of this process can be seen from the

features of patients with xeroderma pigmentosum

(XP). XP patients show a wide variety of skin

abnormalities on sun-exposed areas including freck-

ling, hypo- and hyperpigmentation and ultimately

multiple skin cancers. The incidence of skin cancer

in XP individuals has been estimated to be 1000-fold

higher than that in unaffected individuals. In the

majority of XP patients this hypersensitivity to the

effects of sunlight results from a genetic deﬁciency in

NER. In , 20% of XP individuals however, NER is

normal. These so-called XP variants (XPV) are

deﬁcient in their ability to produce intact daughter

DNA strands during DNA replication after exposure

of the cells to UV-irradiation. In other words, they are

defective in their ability to replicate DNA damaged by

ultraviolet light.

Pol

and the Y-Family of

DNA Polymerases

Although the cellular deﬁciency in XPV was identiﬁed in

1975, the molecular basis for the defect was not

discovered until 1999. The gene defective in XPV cells

was found to encode a new DNA polymerase designated

DNA polymerase

or pol

. Pol

is a protein of 713

amino acids (aa) with no sequence similarity to

previously discovered DNA polymerases, but it does

share sequence similarity with several other DNA

polymerases that were discovered at about the same

time. This new group of polymerases was designated the

“Y-family.” They show sequence conservation over

a region of , 400 aa, which contains the catalytic

domain, and usually forms the N-terminal part of the

protein. Y-family polymerases have a C-terminal exten-

sion, which is not conserved between family members.

CATALYTIC ACTIVITY

DNA polymerases that carry out the replication of

undamaged DNA are very efﬁcient and accurate, but

they are blocked by most types of DNA damage

including both major UV photoproducts. In contrast,

pol

is a rather poor polymerase on undamaged DNA.

It dissociates after incorporating a few nucleotides and

it is rather error-prone. However pol

, in contrast to all

other eukaryotic polymerases, is able to synthesize past

a T-T CPD with the same efﬁciency as past undamaged

T’s. Furthermore in most instances the “correct” bases,

namely two A’s are inserted opposite the T’s of the

CPD. The reason for these properties became clear

when the catalytic domains of several members of

the Y-family (including yeast pol

) were crystallized

and their three-dimensional structures solved. Despite

the lack of sequence similarity between classical and

Y-family polymerases, the three-dimensional structures

did have the three subdomains, designated ﬁnger,

thumb and palm, found in all other DNA polymerases,

with the Y-family having an extra subdomain designated

pad or little ﬁnger. However the classical polymerases

form a closed tight structure, which only permits

normal bases into the active site. In contrast, pol

has

a much more open structure with two important

consequences. Firstly, it has a relatively low stringency,

so that incorrect bases can be incorporated rather

easily. Secondly the open structure is able to accom-

modate the CPD in the template strand and the

structure ensures that in general the correct bases are

inserted opposite the bases in the CPD. Thus pol

tailor-made for replicating past CPDs. It can insert the

appropriate bases opposite the damage and extend

from these bases, but then it will soon dissociate from

the template, so that the more accurate replicative

polymerases can take over again. The ability to bypass

DNA damage is termed translesion synthesis (TLS) and

is shown schematically in Figure 1.

Apart from the CPD, pol

is able to replicate past

some other types of damage in vitro, but in most cases, it

is much less efﬁcient with other types of damaged

templates than with undamaged or CPD-containing

DNA. It is not yet known if it is involved in bypassing

any of these other types of damage in vivo.

The importance of pol

is seen in the severe clinical

characteristics of XPV patients and in the very high UV-

induced mutation frequency in XPV cells. The UV-

mutability of XPV cells is several-fold higher than that of

normal cells and the frequency of all types of mutations

is increased. This is evidence that in the absence of pol

something else is able to do its job, but the replacement

is less efﬁcient and makes more mistakes.

LOCALIZATION

The catalytic domain of pol

is contained within the ﬁrst

430 aa. What then is the function of the C-terminal

280 aa? DNA replication takes place during the S phase

of the cell cycle in “replication factories” inside the cell

nucleus. These factories can be visualized either by

immuno-ﬂuorescence with an antibody to a replication

protein, or by tagging proteins involved in DNA

replication with the green ﬂuorescent protein (GFP).

The factories appear as bright nuclear foci when viewed

by ﬂuorescence microscopy. Pol

also localizes in

replication factories during DNA replication, so that it

is always on hand in case the replication machinery

encounters DNA damage (Figure 2). It is the C-terminal

120 aa of pol

(aa 595–713) that are responsible for

this localization into replication factories. This C-

terminal domain contains a nuclear localization

sequence that is required for transporting pol

into the

nucleus, as well as a C

zinc ﬁnger and a sequence

found in proteins that bind to the replication accessory

protein, PCNA. Both of these motifs are needed to

localize pol

in replication factories. The function of aa

430–595 in pol

has not yet been determined but it is

doubtless involved in interactions with other proteins.

The domain structure of pol

is summarized in Figure 3.

FIGURE 1 Translesion synthesis by pol

. Replicative DNA synthesis is blocked by a CPD (indicated by red blocks). This leads to a switch to pol

(step 1), which replicates past the CPD (step 2) and then dissociates, so that normal replication can restart (step 3).

FIGURE 2 Localization of pol

in replication foci. Pol

, tagged with

green ﬂuorescent protein, localizes into nuclear foci during DNA

replication.

420 XPV DNA POLYMERASE AND ULTRAVIOLET DAMAGE BYPASS

Mutations in XPV Patients

Mutations have been identiﬁed in the pol

gene in some

35 patients. The majority of the mutations result in

truncations within the catalytic subunit and would not

be expected to result in any functional pol

. This

suggests that pol

is not an essential protein, since its

absence is compatible with life. Several mis-sense

mutations within the catalytic domain have also been

identiﬁed and they are predicted either to inhibit

binding to DNA or to interfere with the 3D structure

of the protein. A small number of mutations do not

affect the catalytic domain, but truncate the protein

close to the C terminus. These mutant proteins would

be expected to be fully active, but to lack the

localization domain in the C terminus, which is

required for transporting the polymerase into the

nucleus. There is no clear relationship between the

site or type of mutation and the severity of the clinical

features, even though the severity of features differs

widely between patients.

Replication Past Other Types

of UV Damage

Although the CPD is the major UV photoproduct, the 6-

4 PP is also formed in substantial quantities (, 1/3 the

frequency of the CPD). No single polymerase has been

found that is able to carry out TLS past a 6-4 PP, which

produces a much greater distortion in the DNA than a

CPD. However two polymerases acting in concert are

able to accomplish this. Insertion of the nucleotides

opposite the 6-4 PP can be carried out either by pol

its paralogue, pol

, but neither of these can extend

further from the incorporated nucleotides. However

once nucleotides have been inserted opposite

the damaged bases, they can be extended by DNA

polymerase

. So far, evidence for this dual polymerase

mechanism has only been obtained in vitro. It is

likely that this mechanism is also used in vivo, but as

yet it is not known which polymerase carries out the

insertion step.