Postpartum maternity leave may have a positive effect on breastfeeding among full-time workers, particularly those who hold nonmanagerial positions, lack job flexibility, or experience psychosocial distress. Pediatricians should encourage patients to take maternity leave and advocate for extending paid postpartum leave and flexibility in working conditions for breastfeeding women.

We developed an in vivo selection to identify 3-azido-3-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase ␤ (pol ␤). The selection utilizes pol ␤'s ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol ␤ allows SC18-12 cells to grow, but they depend on pol ␤ activity, so inhibition of pol ␤ by AZT kills them. We screened a library of randomly mutated pol ␤ cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase. The substitutions D246V and R253M result in reductions in the steadystate catalytic efficiency (K cat /K m ) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance. The R253M enzyme exhibits significantly higher K m (dTTP) and K cat (dTTP) values, implying that the incorporation reaction is altered. These are the first pol ␤ mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.Efficient and accurate synthesis of DNA, during replication and repair, is essential to the integrity of any genome. Template-directed synthesis requires that a polymerase select the appropriate deoxynucleotide triphosphate (dNTP) and exclude incorrect bases. The interaction between a polymerase and substrates must therefore be highly specific, yet flexible, in order to maintain sequence fidelity. The smallest of the eukaryotic enzymes that accomplish this reaction is DNA polymerase ␤ (pol ␤), 1 a mammalian polymerase which fills short gaps in DNA (1). pol ␤ has been implicated in base excision repair (2, 3) and meiosis (4). pol ␤ is highly suitable for structure-function studies of the molecular mechanism of DNA synthesis due to the availability of information on the polymerase-DNA-substrate ternary complex (5, 6).DNA synthesis by pol ␤ can be compromised by the nucleoside analog drug AZT, which closely resembles a normal substrate. AZT-triphosphate (AZT-TP) presents a normal thymine moiety which may form a Watson-Crick base pair with adenosine, but has a modified sugar ring that results in chain termination. pol ␤ incorporates AZT into DNA in vitro (7) implying that pol ␤ is not able to distinguish perfectly between the drug and the natural nucleotide substrate. This susceptibility of pol ␤ makes AZT resistance a useful probe of enzyme-substrate interactions involved in DNA synthesis.The molecular basis for polymerase substrate specificity, and specifically AZT discrimination, is not well understood. The clinical problem of AZT-resistant HIV has been attributed to mutations in HIV reverse transcriptase. However, the mutant reverse transcriptase enzymes have exhibited little or no change in AZT-TP incorporation...

The LIM motif is a cysteine- and histidine-rich sequence that was first identified in proteins involved in control of gene expression and cell differentiation. In order to characterize structural features of the LIM domain, we have carried out biophysical studies on two polypeptides that display LIM domains: the cysteine-rich intestinal protein (CRIP) and a fragment of the cysteine-rich protein (CRP). Bacterial expression vectors were constructed for the intact CRIP molecule and the C-terminal half of CRP, designated LIM2, such that each expressed protein contained a single LIM domain. Both proteins were recovered as soluble, Zn(II)-containing proteins. The metal coordination properties of these two distinct LIM domain proteins were highly similar, suggesting that a common structural architecture may exist in LIM domain proteins. Both proteins exhibit a maximum of two tetrahedrally bound Zn(II) ions per molecule. Electronic spectroscopy of Co(II) complexes and 113Cd NMR of Cd(II) complexes of CRIP and LIM2 revealed a similar ligand field pattern with one tetrathiolate (S4) site and one S3N1 site for divalent metal ions. The nitrogen ligand was shown to arise from a histidyl imidazole by heteronuclear multiple quantum coherence NMR. The eight conserved residues within the LIM domains of CRIP and LIM2 include seven cysteines and one histidine. It is likely that these conserved residues generate the S4 and S3N1 Zn(II)-binding sites. Metal binding to the two sites within a single LIM domain is sequential, with preferential occupancy of the S4 site. Slow metal ion exchange occurs between sites within an LIM domain, and metal exchange with exogenous metal ions is observed, with exchange at the S3N1 site being kinetically more facile. In the absence of metal binding both proteins appear to be substantially unfolded. Metal binding stabilizes a tertiary fold containing appreciable secondary structural elements. The common metal ion coordination in CRIP and LIM2 suggests that the LIM motif may constitute a structural module with conserved features.

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The DNA polymerase ␤ mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol ␤ wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase ␤ is critical for mispair extension fidelity. DNA polymerase ␤ (pol ␤)1 is a mammalian polymerase that fills short gaps in DNA (1, 2). pol ␤ has been implicated in base excision repair (3, 4) and appears to function during meiosis (5).Homozygous deletion of the pol ␤ gene in mice is an embryonic lethal event (6), indicating that pol ␤ activity is absolutely required during development, although its specific role is not clear. The best characterized function of pol ␤ is gap-filling synthesis during base excision repair (BER). The BER pathway repairs oxidative and alkylation damage, as well as other DNA lesions. After excision of a damaged base by a DNA glycosylase, AP endonuclease (APE) binds to the abasic site and recruits pol ␤ into a complex with the APE and DNA (7). APE incises the DNA, and pol ␤ conducts both the gap-filling synthesis and the deoxyribophosphatase steps (1,8,9). As AP sites are believed to occur in eukaryotic cells about 10,000 times per cell per day (10), this repair pathway is vital for genomic stability.pol ␤ has also been implicated in the process of meiotic recombination. Plug et al. (5) found that pol ␤ dynamically localizes to the synaptonemal complexes formed by chromosome pairs during meiosis. For this reason, pol ␤ is suspected of participating in meiosis.pol ␤ may also have a role in DNA replication. It is required for conversion of single-stranded to double-stranded DNA in Xenopus extracts (11) and is able to join Okazaki fragments (...

Little is known about modifiable lifestyle factors beyond quitting smoking that could prevent preterm delivery (PTD, <37 weeks gestation). We examined the individual and joint associations of pre-pregnancy BMI, second trimester exercise and sleep on PTD. We conducted a nested, population-based case-control study interviewing postpartum 344 cases delivering at <37 weeks, as identified by clinical estimate of gestational age from prenatal screening records, and 698 term controls, excluding term low birthweight. Eligible women participated in California's statewide Prenatal Screening Program, worked during pregnancy, and delivered a singleton birth in Southern California in 2002-2003. Modeled separately, moderate (odds ratio [OR] = 0.90; 95% confidence interval [CI] = 0.84-0.96--per hour/week) and vigorous (OR = 0.67; 95% CI = 0.46-0.98 for yes vs. no) exercise during the second trimester were associated with a reduced odds of PTD, and sleep duration was not (OR = 1.09, 95% CI = 0.80-1.48 for <7 h; OR = 0.88, 95% CI = 0.57-1.48 for >8 h vs. 7-8 h). When sleep and exercise variables were modeled together along with pre-pregnancy BMI, only moderate exercise (OR = 0.91; 95% CI 0.85-0.98) continued to be associated with reduced odds of PTD. The benefits of moderate exercise appeared strongest for women with BMI greater than 24 kg/m(2) (OR = 0.85; 95% CI = 0.79-0.93) and weakened with decreasing BMI. No other interactions were found. Moderate exercise is associated with reduced PTD, particularly for women with BMI above the normal range. The results are of public health relevance given that these risk factors are potentially modifiable both pre-conceptionally and during pregnancy and rates of PTD are still high in the United States.

Endogenous DNA damage occurs at a rate of at least 20,000 lesions per cell per day. Base excision repair (BER) is a key pathway for maintaining genome stability. Several pol β variants were identified as conferring resistance to 3′-azido-3′-deoxythymidine (AZT) in E. coli.(1) Detailed biochemical studies on one of these AZT resistant variants, His285 to Asp, have shown that the H285D variant of pol β possesses presteady-state kinetics that are similar to the wild-type polymerase. In gap filling assays with 5-bp gapped DNA, H285D showed a slight mutator phenotype. In depth single turnover kinetic analysis revealed that H285D is much more efficient than wild-type pol β at extending mispaired primer termini. This mispair extension property of H285D is attributed to a greatly increased binding to the next correct nucleotide in the presence of a mispair. This change in K d(dNTP),app is not accompanied by a change in k pol ; values for k pol are the same for both H285D and wild-type. Close examination of available structural data, as well as molecular modeling has shown that residue 285 is able to make several stabilizing contacts in the fingers domain of the polymerase and the introduction of a negatively charged side chain could have important effects on the enzyme. It is postulated that the loss of the contact between His285, Lys289, and Ile323 is responsible for the ability of H285D to extend mispairs through disruption of contacts near the Cterminal end of pol β and propagation into the nucleotide binding pocket.Endogenous DNA damage occurs at a rate of at least 20,000 lesions per cell per day (2). These endogenous lesions are repaired by the base excision repair (BER) machinery, and their correct repair is critical for genome stability. A key enzyme in the BER pathway is DNA polymerase beta (polβ), which removes the deoxyribose phosphate group (dRP) and fills in the gap with a nucleotide after the DNA lesion is excised. A relatively error-prone polymerase, pol β inserts an incorrect nucleotide in approximately one out of every 10,000 nucleotide insertions (3). Given that BER is responsible for repairing about 20,000 DNA lesions per cell per day, pol β could potentially incorporate incorrect nucleotides on the order of one per cell per day. These errors in DNA repair can be carried through further cell divisions, which can increase genomic instability. Increased genomic instability has been shown to lead to the generation of a mutator phenotype, which in turn can produce a malignant phenotype (4).In BER, damaged DNA bases are recognized and excised by a specific DNA glycosylase, which generates a mutagenic apurinic/apyrimidinic (AP) site (5). Next, AP Endonuclease 1 incises the DNA backbone on the 3′ side of the AP site, leaving behind a dRP group (6), which §To whom correspondence should be addressed. E-mail: joann.sweasy@yale.edu, Department of Therapeutic Radiology and Genetics. Yale University School of Medicine. New Haven, CT 06520, Tel.: 203-737-2626, Fax: 203-785-6309. NIH Public Access is then ...

The relation of CRH with PTD is moderated by exposure to chronic stressors during pregnancy.